Methods of Reducing Teratoma Formation During Allogeneic Stem Cell Therapy

ABSTRACT

The present application relates to methods and compositions for treating diseased or damaged cardiac tissue comprising regenerative cells harvested from donor cardiac tissue. In one embodiment, regenerative cells are harvested from an allogeneic source and after administration result in increased viability and/or functional improvement of damaged or diseased cardiac tissue.

RELATED CASES

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 11/666,685, filed Apr. 21, 2008, which is the U.S.National Phase application under 35 U.S.C. §371 InternationalApplication No. PCT/US2005/040359 filed on Nov. 8, 2005, which claimsthe benefit of U.S. Provisional Application No. 60/625,695 filed Nov. 8,2004, the disclosures of each of which are expressly incorporatedherein.

STATEMENT REGARDING GOVERNMENT SPONSORED GRANT

The studies disclosed herein were made with Government support under oneor more of the National Institutes of Health Research Project GrantsHL095203 HL103356, HL081028, and HL083109. The United States Governmenthas certain rights in this invention.

BACKGROUND

1. Field of the Invention

Several embodiments of the present application relate generally toregenerative cells, methods of preparing regenerative cells, andcompositions comprising regenerative cells for use in transplant forrepair of damaged tissue. In one embodiment, regenerative cells isolatedfrom donor heart tissue may be cultured, expanded, and administered to arecipient in order to repair damaged cardiac tissue of the recipient.

2. Description of the Related Art

Stem cells are characterized by the ability to renew themselves throughmitotic cell division and the ability to differentiate into a diverserange of specialized cell types. The two primary types of mammalian stemcells are embryonic stem cells and adult stem cells (i.e., non-embryonicstem cells). Embryonic stem cells are isolated from the inner cell massof blastocysts and are pluripotent, meaning that the cells have thecapacity to differentiate into all of the specialized embryonic tissues.Adult stem cells are isolated from adult tissues and function as anongoing repair system for adult organs.

Coronary heart disease is presently the leading cause of death in theUnited States, taking more than 650,000 lives annually. According to theAmerican Heart Association, 1.2 million people suffer from a heartattack (or myocardial infarction, MI) every year in America. Of thosewho survive a first MI, many (25% of men and 38% of women survivors)will still die within one year of the MI. Currently, 16 millionAmericans are MI survivors or suffer from angina (chest pain due tocoronary heart disease). Coronary heart disease can deteriorate intoheart failure for many patients. 5 million Americans are currentlysuffering from heart failure, with 550,000 new diagnoses each year.Regardless of the etiology of their conditions, many of those sufferingfrom coronary heart disease or heart failure have suffered long lastingand severe heart tissue damage, which often leads to a reduced qualityof life.

SUMMARY

Given the vast potential of stem cell therapy to revolutionize medicaltreatment, there exists a need in for effective and efficientadministration of stem cells, compositions comprising stem cells, orderivatives of stem cells to a recipient in order to elicit therapeuticeffects, including, among others, tissue regeneration. In particular, inthe context of coronary heart disease, there is a need for improvedmethods to isolate, prepare and administer cell-based compositions torecipients in order to ameliorate and/or treat the cardiac tissue damagethat results from adverse cardiac events associated with coronary heartdisease.

In several embodiments of the invention, a method of treating an adversecardiac event (such as myocardial infarction) is provided, wherein themethod comprises obtaining a cardiac biopsy sample from a first patient,culturing the sample to obtain regenerative cells, and implanting theregenerative cells into a second patient. This type of allogeneictransplant, according to several embodiments, is particularlyadvantageous because the regenerative cells do not evoke a significantchronic immune response that is adverse to the patient. Instead, theregenerative cells trigger a cascade of therapeutic signaling effects(e.g., a paracrine effect) prior to destruction via an acute immuneresponse that destroys the regenerative cells. In this manner, accordingto several embodiments, “off-the-shelf” regenerative cells can beproduced to treat patients suffering from cardiac diseases. Thus thepatient need not have healthy tissue from which to harvest his or herown cells (as is the case for an autologous transplant). Moreover, evenwhen a patient has healthy heart tissue for biopsy, the patient need nothave to wait for the culturing process. Instead, the “off-the-shelf”allogeneic cells may be available with little or no time delay.

According to several embodiments, methods for increasing function of adamaged or diseased heart of a mammal are provided. In one embodiment, apopulation of cells is administered to the mammal, wherein thepopulation of cells increases cardiac function in the mammal. Thepopulation of cells, in one embodiment, is obtained by the process ofculturing cells obtained from cardiospheres on a surface as a monolayer.In several embodiments, a population of in vitro-expanded cells isadministered to the mammal. In some embodiments, the cells have thecapacity to form cardiospheres in suspension culture. According to oneembodiment, the cells are not, however, in the form of cardiosphereswhen administered.

Methods for treating a mammal with a damaged or diseased heart areprovided which comprise, in some embodiments, obtaining heart tissuefrom the damaged or diseased heart of the mammal. In severalembodiments, heart tissue is obtained from a heart of a donor. In someembodiments, the heart tissue is obtained by way of a percutaneousendomyocardial biopsy. In some embodiments, the heart tissue is treatedto obtain and expand a population of cardiac stem cells and the cardiacstem cells and/or their progeny are subsequently introduced into thedamaged or diseased heart.

In some embodiments, methods are provided for treating a mammal with adamaged or diseased organ (e.g., not necessarily the heart). In oneembodiment, tissue is obtained from the damaged or diseased organ of themammal or from a healthy organ of a donor by, for example, apercutaneous biopsy. In several embodiments, the tissue is treated toobtain and expand a population of stem cells. The stem cells and/ortheir progeny are introduced into the damaged or diseased organ of themammal. In one embodiment, a method of treating kidney damage isprovided. In one embodiment, a kidney biopsy specimen is incubated inthe presence of a protease. The cells liberated from the biopsy specimenby the protease incubation are collected. The collected cells arecultured on a surface as a monolayer to expand number of cells, whichare then introduced to the damaged kidney.

In several embodiments, cells (e.g., stem cells) are obtained fromallogeneic donor tissue, including but not limited to donated organshaving tissue that is at least partially healthy and harvestable. Insome embodiments, cardiac tissue is obtained from hearts deemedunsuitable for transplantation. Accordingly, in several embodiments, themethods provided herein are particularly advantageous because theyutilize donor hearts that would otherwise have been discarded orunder-utilized.

In some embodiments, methods for treating tissue (including but notlimited to a cardiac biopsy specimen) are provided. In severalembodiments, the tissue is incubated in the presence of a protease. Thecells that are liberated from the tissue by the protease incubation arecollected. In some embodiments, the collected cells are cultured on asurface as a monolayer to expand number of cells.

In some embodiments, methods for expanding a population of cells(including but not limited to cardiac stem cells) are provided. Inseveral embodiments, one or more bodies (e.g., cardiospheres) aredisaggregated to individual cells or smaller aggregates of cells. Theindividual cells or smaller aggregates of cells are cultured on asurface as a monolayer, in some embodiments. In one embodiment, apopulation of in vitro-expanded cells in a monolayer is provided. Thecells have the capacity to form cardiospheres in suspension culture. Thecells are not, however, in the form of cardiospheres in someembodiments. In still another embodiment, a population of cells made bythe process of culturing cells on a surface as a monolayer is provided.In some embodiments, the cells are obtained from disaggregatedcardiospheres.

Although several embodiments of the invention are used for autologousadministration, many embodiments are suitable for allogeneicadministration. Allogeneic administration is advantageous in severalembodiments because it is readily available for immediate administrationto patients.

Use of certain types of cells for cellular therapy may be hampered bythe unwanted differentiation and growth of administered cells into celltypes that are distinct (and in some cases not functionallycomplementary) to the target tissue. Teratoma formation is thus apotential concern with certain types of cell therapy. To address suchconcerns, in several embodiments, there is provided a method for thereduction of teratoma formation following the delivery of non-self cellsto a first subject (e.g., cells isolated from a tissue source harvestedfrom a second subject, wherein the second subject is an adult)comprising delivering to a first subject a population of regenerativecells, wherein the at least a portion of the regenerative cells engraftinto a target tissue of the first subject after delivery to the subject,wherein the regenerative cells express one or more factors that reduceteratoma formation (e.g., in comparison to the delivery of embryoniccells to a subject). In some embodiments, it is the engraftment of theregenerative cells that reduces teratoma formation, at least in part dueto the retention of the cells at the desired target site. In someembodiments, the period of engraftment reduces teratoma formation. Forexample, in some embodiments the period of engraftment is short term(e.g., a few days to several weeks) and insufficient for teratomaformation to occur. In some embodiments, whether engrafted or not, deathand/or destruction of the regenerative cells reduces teratoma formation.In several embodiments, it is the combination of two or more ofengraftment of the cells, the period of engraftment, the destruction ofthe cells, and/or the factors released by the cells that reducesteratoma formation.

In several embodiments, there is provided a method of treating a firstsubject having damaged cardiac tissue with allogeneic cells from asecond subject, the method comprising obtaining a plurality ofregenerative cells (e.g., CDCs) harvested from the cardiac tissue of asecond subject, wherein the administered CDCs generate one or morecytokines, chemokines or diffusible factors, wherein, afteradministration, at least a portion of the administered CDCs engraft intothe cardiac tissue of the first subject; and wherein the one or moregenerated cytokines, chemokines or diffusible factors or the engraftmentimproves the function of the damaged cardiac tissue, thereby treatingthe first subject. In several embodiments, the cells have been expandedin culture to yield a population of cardiosphere-derived cells (CDCs).In one embodiment, the CDCs are not pluripotent and are committed todifferentiating into cardiac tissue, thereby reducing the risk ofproducing undesired tissue growth.

In several embodiments, the engraftment of administered cells persistsfor a time period ranging from about 1 week to about 6 weeks. In severalembodiments, during the period of engraftment at least a portion of theregenerative cells are destroyed by the immune system of the firstsubject. In several embodiments, the destruction of cells by the immunesystem is, at least in part, responsible for the reduced teratomaformation as a result of the reduced residence time of the cells.

In several embodiments, during the period of engraftment, theregenerative cells induce endogenous cells to express one or morefactors that reduce teratoma formation. Thus, in some embodiments, thecombination of factors generated from the regenerative cells and thefactors induced to be generated by the endogenous cells is responsiblefor the reduction in teratoma formation. In several embodiments,expression of the factors comprises cell-surface expression. In severalembodiments, expression of the factors comprises release of the factorsfrom the cells.

In several embodiments, the delivery of the regenerative cells is forthe purpose of repairing a damaged or diseased tissue of the firstsubject. In several such embodiments, the damaged or diseased tissue ofthe first subject comprises damaged or diseased cardiac tissue. In someembodiments, the population of regenerative cells comprises cardiac stemcells. In several embodiments, the cardiac stem cells are selected fromthe group consisting of cardiospheres, cardiosphere-derived cells, and asubsequent generation of cardiospheres.

In several embodiments, the regenerative cells express one or more stemcell markers selected from the group consisting of c-kit, CD90, andsca-1. In several embodiments, the regenerative cells express one ormore endothelial cell markers selected from the group consisting of KDR,flk-1, CD31, von Willebrand factor, Ve-cadherin, and smooth muscle alphaactin. In several embodiments, the regenerative cells express one ormore of the stem cell markers or one or more of the endothelial cellmarkers, but are not selected for, enriched, purified or otherwisepreferentially obtained based on the expression of the one or moreexpressed markers. In several embodiments, the isolated regenerativecells are expanded in culture prior to delivery. In several embodiments,the culturing of the cells is performed in order to induce theexpression of one or more of the markers above.

In several embodiments, the isolated regenerative cells generateteratoma-reducing factors in culture. In one embodiment, the methodfurther comprises isolating the teratoma-reducing factors from theculture. In one embodiment, the method also comprises delivering theisolated teratoma-reducing factors from the culture to the firstsubject. In one embodiment, delivery of the isolated teratoma-reducingfactors is prior to delivery of the regenerative cells. In oneembodiment, delivery of the isolated teratoma-reducing factors isconcurrent with delivery of the regenerative cells. In one embodiment,delivery of the isolated teratoma-reducing factors is after delivery ofthe regenerative cells. In several embodiments, delivery of the isolatedteratoma-reducing factors is at multiple time points throughout theperiod of engraftment of the regenerative cells. In several embodiments,the delivery of factors isolated in culture supplements the expressed(and/or the induced) generation of teratoma-reducing factors.

In one embodiment, between about 1×10⁶ and about 100×10⁶ of the CDCs, orthe regenerative cells, are administered to first subject.

In several embodiments, there is provided a method of treating a firstsubject having diseased or damaged cardiac tissue with allogeneicregenerative cells obtained from a second subject, the method comprisingobtaining a plurality of regenerative cells for administration to afirst subject and administering at least a portion of the population ofexpanded regenerative cells to the first subject.

In several embodiments wherein the regenerative cells are harvested fromcardiac tissue obtained from a second subject and subsequently expandedin culture to yield a population of expanded regenerative cells, atleast a portion of which are suitable for administration. In severalembodiments, after administration, at least a portion of theadministered regenerative cells is destroyed by the first subject'simmune system. However, in some embodiments, the administeredregenerative cells generate one or more paracrine signalspost-administration and prior to the destruction. In severalembodiments, the one or more paracrine signals improve one or more ofthe viability or function of the damaged cardiac tissue, therebytreating the first subject. In one embodiment, the regenerative cellsare cardiosphere-derived cells (CDCs). In one embodiment, theregenerative cells are cardiospheres. In one embodiment, a mixture ofCDCs and cardiospheres is used.

In several embodiments, the regenerative cells are harvested fromcardiac tissue obtained from a second subject and subsequently expandedin culture to yield CDCs. In some embodiments, after administration, atleast a portion of the CDCs is destroyed by the immune function of thefirst subject. In several embodiments, the administered CDCs generateone or more paracrine signals post-administration and prior to thedestruction. In several embodiments, the one or more paracrine signalsimprove one or more of the viability or function of the damaged cardiactissue, thereby treating the first subject.

In several embodiments, the viability or function of the damaged cardiactissue is improved directly by the paracrine signals. In severalembodiments, the viability or function of the damaged cardiac tissue isimproved by an indirect mechanism induced by the paracrine signals. Insome embodiments, the indirect mechanism comprises recruitment ofendogenous cells that repair cardiac tissue. In some embodiments, theindirect mechanism comprises induction of production of paracrinefactors by endogenous cells. As a result, in some embodiments, a feedforward repair cascade is initiated, wherein administered cells andtheir paracrine signals induce further paracrine signal generation byendogenous cells, and effect a more robust repair (e.g., viability orfunction) of cardiac tissue.

In several embodiments, the administration of the cells results in anincrease in at least one of left ventricular percent fractional area andleft ventricular ejection fraction. In several embodiments, increases ofat least about 5%, 10%, 15%, or more are realized. In other embodiments(e.g., myocardial infarction) scar tissue formation is reduced. In someembodiments, administration of the cells induces pro-survival paracrinesignals that improve the viability and/or function of the damagedcardiac tissue. Thus, in such embodiments, the administration inducesanti-apoptotic (or other cell death pathways) signals or cascades thatresult in improved viability, despite an injurious event or disease thataffected or is affecting the cardiac tissue. In several embodiments, thepro-survival paracrine signals decrease apoptosis in the damaged cardiactissue. In several embodiments the pro-survival paracrine signalsincrease capillary density in the damaged or diseased cardiac tissue. Insome such embodiments, the increased capillary density increases theflow of oxygenated blood the regions of the cardiac tissue, therebyimproving the viability of the tissue by reducing periods of ischemia,for example.

In several embodiments, the paracrine signals comprise one or moregrowth factors or cytokines. In one embodiment, the growth factors orcytokines comprise one or more of VEGF, HGF, and IGFI. In thealternative or in conjunction with these factors, other growth factorsor cytokines are released (or presented on the surface) by the cells, inother embodiments.

In several embodiments the destruction of the cells is accomplished viaphagocytosis. In some embodiments, immune responses (humoral orcomplement mediated) act to destroy the regenerative cells. In severalembodiments, natural death of the administered cells (e.g., apoptosis)occurs, thereby accounting for destruction of the administered cells.

In several embodiments, the cells express one or more stem cell markersselected from the group consisting of: CD105, c-kit, CD90, and sca-1 andone or more endothelial cell markers selected from the group consistingof KDR, flk-1, CD31, von Willebrand factor, Ve-cadherin, and smoothmuscle alpha actin. In some embodiments, other cardiac, vascular, orendothelial markers are expressed within or on the cells. In someembodiments, the cells may be selected for by the presence or expressionof certain markers. However, in several embodiments, no selection orenrichment based on marker selection is made.

In one embodiment, the diseased or damaged cardiac tissue is the resultof one or more of acute heart failure (e.g., a stroke or MI) or chronicheart failure (e.g., congestive heart failure). In several embodiments,about 1×10⁵ to about 1×10⁷ of the cells are administered. In severalembodiments, the dose is varied depending on the size and/or age of asubject receiving the cells. In some embodiments (e.g., those thatinduce feed-forward effects in endogenous cells), smaller numbers ofcells are optionally administered. Different routes of administrationare also used, depending on the embodiment. For example, theregenerative cells may be administered by intravenous, intra-arterial,intracoronary, or intramyocardial routes (or other routes) ofadministration.

In one embodiment, there is provided a method of treating a firstsubject having diseased or damaged cardiac tissue with allogeneicregenerative cells obtained from a second subject, the method comprisingobtaining a plurality of regenerative cells for administration to afirst subject, administering the expanded regenerative cells to a firstsubject having damaged cardiac tissue, wherein the regenerative cellsare harvested from cardiac tissue obtained from a second subject andsubsequently expanded in culture to yield the population of expandedregenerative cells, wherein, after administration, at least a portion ofthe expanded regenerative cells engraft into the cardiac tissue of thefirst subject; wherein the administered regenerative cells generate oneor more paracrine signals; and wherein the one or more paracrine signalsimprove one or more of the viability or function of the damaged cardiactissue, thereby treating the first subject. In one embodiment, theregenerative cells comprise cardiosphere-derived cells (CDCs), whereinthe CDCs are about 5 and 20 microns in diameter. In one embodiment, theCDCs are delivered to the first subject via intracoronaryadministration. In some embodiments, other administration routes areused, for example, intravenous, direct myocardial injection, etc.Selection of the optimal administration route is based upon, among otherfactors, dose of cells to be delivered, location of the area of damagedtissue, severity of tissue damage, and the like.

In addition to the methods disclosed above, there is also provided apopulation of allogeneic cells for administration to a subject for therepair of damaged cardiac tissue, comprising cardiosphere derived cells(CDCs) isolated from a first subject, and suitable for administration toa second subject that is allogeneic with respect to the first subject,and expanded in culture.

In several embodiments, there is provided a population of allogeneiccells isolated from a first subject and suitable for administration to asecond subject for the repair of damaged cardiac tissue of the secondsubject comprising cardiosphere derived cells (CDCs) isolated from afirst subject and expanded in culture, wherein the CDCs express the stemcell markers CD105 and c-kit, but are not screened, subfractionated, orotherwise selected based on the expression of the markers, wherein theCDCs express one or more products that improve the viability orfunctionality of the damaged cardiac tissue of the second subject,

In several embodiments, the CDCs have a diameter of between about 5 and20 microns. In some embodiments, the size of the CDCs is advantageous inthat a greater variety of delivery routes are available with reducedrisk of inducing embolization of the microcirculation uponadministration. In several embodiments, administration of the CDCs to asecond subject results in engraftment of the CDCs in the cardiac tissueof the second subject for at least about 3 weeks. During that period ofengraftment, in several embodiments, the CDCs generate one or moreparacrine signals (or expressed products) that yield improvements in theviability or function of the damaged cardiac tissue. In severalembodiments, generation of paracrine factors persists beyond the periodof engraftment (e.g., the administered cells induce a cascade of eventsthat results in the propagation of paracrine signal production, even inthe absence of some or all of the originally administered cells). Asdiscussed above, in several embodiments, the administered cells induceparacrine factor production in endogenous cells.

In several embodiments, the CDCs express a variety of markers thatidentify the cell types that comprise a CDC population. In severalembodiments, the CDCs express the stem cell marker CD105. In severalembodiments, the CDCs further express one or more stem cell markersselected from the group consisting of: c-kit, CD90, and sca-1. Inseveral embodiments the CDCs further express one or more endothelialcell markers selected from the group consisting of: KDR, flk-1, CD31,von Willebrand factor, Ve-cadherin, and smooth muscle alpha actin. Asdiscussed herein, in several embodiments, while the CDCs express one ormore of the stem cell markers and/or one or more of the endothelial cellmarkers, the cells are screened, subfractionated, or otherwise selectedfor based on the expression of the one or more expressed markers.

The beneficial effects of the administration of cells, as disclosedherein, is attributable to the cells themselves, one or more of theparacrine factors produced by the cells, or combinations thereof. Inseveral embodiments, the paracrine signals comprise one or more growthfactors or cytokines. In several embodiments, the growth factors orcytokines comprise one or more growth factors or cytokines selected fromthe group consisting of: ENA-78, G-CSF, GM-CSF, GRO, GRO-alpha, I-309,IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-13, IL-15, interferon gamma, MCP-1, MCP-2, MCP-3, M-CSF, MDC,MIG, MIP-1 beta, MIP-1 delta, RANTES, SCF, SDF-1, TGF-beta 1, TNF-beta,EGF, IGF-1, angiogenin, oncostatin M, thrombopoeitin, VEGF, PDGF-BB,leptin, BDNF, BLC, Ck beta 8-1, eotaxin, eotaxin-2, eotaxin-3, FGF-4,FGF-6, FGF-7, Flt-3 ligand, fractalkine, GCP-2, GDNF, HGF, IGFBP-1,IGFBP-2, IGFBP-3, IGFBP-4, IL-16, IP-10, LIF, LIGHT, MCP-4, MIP-3 alpha,NAP-2, NT-3, NT-4, osteopontin, osteoprogenerin, PARC, PIGF, TGF beta 2,TGF beta 3, TIMP-1 and TIMP-2. In several embodiments, the growthfactors or cytokines comprise one or more of VEGF, HGF, and IGFI.

The beneficial effects of administration of the population of allogeneicCDCs are multi-fold, and include both functional improvements andimprovements in viability of the damaged, diseased, and/or surroundingcardiac tissue. In some embodiments, administration of the CDCs resultsin at least a 5%, at least a 10%, or at least a 15% improvement in leftventricular function. In some embodiments, the improvement in leftventricular function persists for at least about 6 weeks afteradministration of the CDCs. In some embodiments, longer term functionalimprovement is achieved (e.g., 2-3 months, 3-5 months, or greater). Insome embodiments, functional improvement is essentially indefinite(e.g., the treatment methods disclosed herein are one-time methods inthat additional doses of cells are not required). However, in someembodiments, a serial dosing regimen is preferred. In severalembodiments, administration of the CDCs results in at least a 2-foldimprovement cardiac cell viability. In some embodiments, similar to thefunctional improvements, the increased viability is also for longerperiod (e.g., the damaged cells live longer and/or endogenous cells livelonger). In some embodiments, the increased viability is due to areduction in apoptosis. In some embodiments, apoptosis still occursnaturally, but cells have an altered threshold for committing to anapoptotic pathway (e.g., a greater degree of damage or longer period ofischemia is required). As discussed herein, in several embodiments, thedamaged cardiac tissue of the first subject is a result of myocardialinfarction, chronic ischemia, or congestive heart disease. In some suchembodiments wherein an infarct caused the damage, administration of theCDCs results in a decrease in infarct size. In some embodiments, thereduction in infarct size ameliorates the increased cardiac function (agreater amount of functionally contractile cardiac tissue) and/or theviability (reduction in scar size allows for better overall tissueperfusion and better tissue viability).

In some instances, damage or disease affects cardiac tissue to such adegree that cardiac function cannot be maintained at a level sufficientto support the continued viability of a subject. There is thereforeprovided a method of supplementing the cardiac function of a firstsubject, the method comprising obtaining a plurality of cardiac stemcells from a second subject for administration to a first subject, andadministering the cardiac stem cells to the first subject. In severalembodiments, the first subject has reduced cardiac function due todamaged cardiac tissue. In some embodiments, the obtained cardiac stemcells are optionally expanded in culture in order to achieve a certainpopulation density prior to administration to the first subject. Inseveral embodiments, the administered cardiac stem cells produce one ormore diffusible factors and the one or more diffusible factors improvethe left ventricular ejection fraction of the heart of the firstsubject, thereby, supplementing the cardiac function of the firstsubject.

In some instances, damage or disease affects cardiac tissue to such adegree that an assist device is required in order to maintain normalcardiac functionality. Even with an assist device (e.g., an implantedpacemaker), in some circumstances, cardiac functionality is still toolow for some individuals. Therefore, there is provided, in severalembodiments, a method of supplementing the function of an implanted leftventricular assist device (LVAD) in a subject, comprising identifying asubject having damaged cardiac tissue and an implanted LVAD anddelivering a plurality of cardiac stem cells to the subject, wherein thedamaged cardiac tissue is functionally assisted by the implanted LVAD.In several embodiments, the plurality of cardiac stem cells is isolatedfrom healthy donor cardiac tissue, wherein the healthy donor tissue iseither from the subject with the LVAD, or preferably from a subject thatis allogeneic to the subject with the LVAD. In several embodiments, atleast a portion of the delivered cardiac stem cells engraft into thedamaged cardiac tissue, although, in some embodiments, at least aportion of the engrafted cardiac stem cells are destroyed by thesubject's immune system. The engrafted cardiac stem cells release one ormore factors prior to the destruction, and as a result, the one or morefactors improve the function of the damaged cardiac tissue, therebysupplementing the function of the LVAD.

In several embodiments, the factors comprise one or more of VEGF, HGF,and IGFI. Other factors, as disclosed herein, are produced by the cells(either the administered cells or the endogenous cells,post-administration) in some embodiments.

In some embodiments, the combination of the direct effect of theengrafted cells (e.g., new healthy functional cardiac cells) with thefactors serves to increase the LVAD supplementation. Moreover, inseveral embodiments, the factors improve the viability of the damagedcardiac tissue. A greater degree of viable cardiac tissue alsoameliorates the functionality of the cardiac tissue as a whole, inseveral embodiments.

In one embodiment, the donor cardiac tissue is allogeneic with respectto the subject. In one embodiment, the donor cardiac tissue isautologous with respect to the subject. In additional embodiments,combinations of allogeneic and autologous cells are used. Suchcombinations may be beneficial in certain patient populations, such asfor example, the extremely immunocompromised. Allogeneic cells producedby the methods herein induce only a limited immune response, if any.However, extremely immunosuppresed individuals may still be susceptibleto minor immune reactions from fully allogeneic cell populations. Thus,combinations of autologous and allogeneic may be used in such cases.

In several embodiments, the cardiac stem cells are selected from thegroup consisting of: cardiospheres, cardiosphere-derived cells (CDCs),and a subsequent generation of cardiospheres. The choice of which celltype is used may be made based on the target location. The size of theCDCs versus the cardiospheres may impact the delivery route and/or thedose delivered. In some embodiments, about 1×10⁵ to about 1×10⁷ cardiacstem cells per kilogram of body weight of the subject are delivered. Insome embodiments, the regenerative cells are delivered by injection. Insome cases, the regenerative cells are delivered during the process ofimplanting an LVAD. In such cases, a simple direct injection may beused. Also, in such cases, the degree of damage or disease (whichcorresponds to the degree of reduced functionality of the heart) can beused to tailor the dose of cells administered. For example, moderatedamage may be primarily addressed by an LVAD, with the administration ofCDCs minorly supplementing the overall cardiac function. In contrast,severe damage may require both the LVAD and the administration of cellsin order to maintain sufficient cardiac function to support theviability of the recipient.

In some embodiments, a subject has an implanted LVAD as a result of atleast one prior myocardial infarction. A single infarction may notrequire the use of a LVAD, depending on its severity. However, asdiscussed above, in several embodiments, even a minor infarction mayoptionally be treated with the cells and methods disclosed herein and/oran LVAD. In some such embodiments, delivery of the cardiac stem cellsreduces the infarct size, which allows a greater supplementation of LVADfunction, as the LVAD is not forced to assist the same amount of stiff,non-contractile scar tissue. Moreover, in several embodiments, thedelivery of the cardiac stem cells increase the left ventricularejection fraction of the subject by about 15%, thereby supplementing thefunction of the LVAD. In some embodiments, greater or lesser increasesin left ventricular function are achieved, which can be tailored to theamount of assistance provided by the LVAD. In some embodiments, however,the administration of cells according to several embodiments hereinnegates the need for an LVAD, as discussed below.

In several embodiments, the damage or disease to a subject's heart is sosevere that the subject is suitable for undergoing a cardiac transplant.In some embodiments, the cardiac stem cells are delivered as a bridge tomaintain left ventricular function until the subject receives a cardiactransplant. In other words, such a great degree of the cardiac tissue isdamaged, diseased, or otherwise compromised, that the administration ofthe cells temporarily maintains cardiac viability and function, untilsuch time as a complete heart transplant can be performed. However, incases with less severe damage, the supplementation of the implanted LVADnegates the need for a cardiac transplant.

In several embodiments, the engrafted cardiac stem cells express one ormore stem cell markers selected from the group consisting of CD105,CD90, c-kit, and sca-1. In some embodiments, the engrafted cardiac stemcells express one or more endothelial cell markers selected from thegroup consisting of KDR, flk-1, CD31, von Willebrand factor,Ve-cadherin, and smooth muscle alpha actin. Despite the wide variety ofmarkers that may be expressed on the cells, e.g., one or more of thestem cell markers CD105 and c-kit, the cells are not screened,subfractionated, or otherwise selected based on the expression of themarkers. In other words the markers are used to characterize the cells,not selectively choose the cells. However, in some embodiments,selection based one or more of such markers is optionally performed.

In some embodiments, autologous cells are administered to patients withleft ventricular dysfunction and a recent myocardial infarction withdelivery occurring by intracoronary infusion via an over-the-wireballoon catheter. In other embodiments, allogeneic cells areadministered to patients undergoing ventricular assist device placement,via intramyocardial injection using a standard needle and syringe and anepicardial approach during LVAD placement. In some embodiments, theallogeneic cells produce an immune response that is similar to theautologous cells.

In several embodiments, regenerative cells (autologous and/orallogeneic) are administered to patients via epicardial injection (orother delivery mechanism) in conjunction with LVAD implantation.

Given the widespread use of LVADs to treat reduced cardiac function,certain subject's that are treated by the methods disclosed herein mayalready have an LVAD implanted. However, there is also provided a methodof reducing the dependence of a subject on an implanted left ventricularassist device (LVAD) comprising administering to the cardiac tissue ofthe subject a population of regenerative cells, wherein, afteradministration, at least a portion of the administered regenerativecells are removed from the subject's cardiac tissue by endogenousmechanisms of the subject, and wherein, prior to the removal, theadministered regenerative cells generate one or more signals that induceimprovement in one or more of the viability or function of the cardiactissue of the subject, thereby reducing the dependence of the subject onthe LVAD.

As discussed herein, certain subjects present with widespread and severecardiac damage and/or decreased functionality as a result of a cardiacinjury (e.g., a myocardial infarction) or disease (e.g., congestiveheart failure). Often, such subjects are likely candidates for hearttransplants. However, the costs, and complications, associated withtransplants mean that such an approach is not a viable solution for allsubjects. There is, therefore, also provided a method of reducing afirst subject's likelihood of having a cardiac transplant, the methodcomprising, obtaining a plurality regenerative cells from a secondsubject that are suitable for administration to the first subject,wherein the regenerative cells are expanded in culture to generate apopulation of regenerative cells prior to administration to the firstsubject, and administering at least a portion of the population ofexpanded regenerative cells to the first subject; wherein theadministered regenerative cells produce one or more diffusible factors,wherein the administered regenerative cells and the one or morediffusible factors induce one or more of stimulation of resident cardiaccells to grow, stimulation of resident cardiac cells to reproduce, orstimulation of resident cardiac cells to improve functionally, therebyreducing the likelihood of the first subject requiring a cardiactransplant.

In some embodiments, the first subject has reduced cardiac function andeither has an LVAD or is a candidate for a heart transplant (or both)due to one or more of an acute ischemic event, chronic ischemia, orcongestive heart disease. In several embodiments, the first subjectreceives cells isolated from the first subject's own tissue (e.g., anautologous transplant). In other embodiments, the first subject isallogeneic with respect to the donor of the tissue from which theregenerative cells are isolated. In several embodiments, theadministration increases the function of the cardiac tissue of the firstsubject thereby reducing the likelihood of the first subject requiring acardiac transplant.

In several embodiments, the regenerative cells are harvested fromhealthy donor cardiac tissue. In several embodiments, the donor cardiactissue is allogeneic with respect to the subject.

In several embodiments, the dose of cells to be administered may bedetermined by virtue of the severity of the cardiac damage or disease,the age of the subject, the subject's overall general health, and otherfactors. However, in several embodiments, about 1×10⁵ to about 1×10⁷regenerative cells per kilogram of body weight of the subject areadministered. In other embodiments, other doses are used. In someembodiments, the regenerative cells are administered by localintramyocardial injection. In some embodiments, the regenerative cellsare administered by epicardial injection. Other routes of administrationare used, in some embodiments, for example, depending on the preciselocation of a LVAD and/or the damaged cardiac tissue. In someembodiments, the regenerative cells are administered during the processof implanting the LVAD. In other embodiments, the regenerative cells areadministered during the process of explanting an implanted LVAD.

While various mechanisms, both direct and indirect, may be involved, insome embodiments, the regenerative cells increase the incidence ofangiogenesis in the subject's cardiac tissue. In some embodiments, theregenerative cells increase the left ventricular ejection fraction ofthe subject by at least about 5%, at least about 10%, at least about15%, or more, thereby reducing the dependence of the subject on theimplanted LVAD.

As discussed herein, in several embodiments, at least a portion of theadministered cells engraft into the target tissue. Additionally, inseveral embodiments, at least a portion of the cells are removed fromthe target tissue by endogenous mechanisms of the subject receiving thecells. In one embodiment, the endogenous mechanisms comprise recruitmentof at least a portion of the subject's immune system. In one embodiment,the endogenous mechanisms comprise induction of apoptosis of theadministered regenerative cells.

In several embodiments, the regenerative cells are selected from thegroup consisting of: cardiospheres, cardiosphere-derived cells (CDCs),and a subsequent generation of cardiospheres. In one embodiment, theregenerative cells are CDCs. In several embodiments, the regenerativecells express one or more of the stem cell markers CD105 and c-kit, butare not screened, subfractionated, or otherwise selected based on theexpression of the markers.

As a result of acute injury to the heart (e.g., a myocardial infarction)or long-term damage there may be an associated increase in cell death asa result, in particular, apoptotic cell death. Therefore, there isprovided a method of decreasing apoptosis in a heart having beenaffected by myocardial infarction, comprising administering a populationof cardiac stem cells to a subject having a heart affected by amyocardial infarction, wherein, after administration, at least a portionof the administered population of cardiac stem cells is destroyed byendogenous mechanisms of the subject, wherein the administeredpopulation of cardiac stem cells generate one or more paracrine signalspost-administration and prior to the destruction, and wherein the one ormore paracrine signals reduce the incidence of apoptosis in the cardiactissue affected by the myocardial infarction.

There is also provided a method of decreasing apoptosis in a hearthaving been affected by myocardial infarction, comprising administeringa population of cardiac stem cells to a subject having a heart affectedby a myocardial infarction, wherein the administered cardiac stem cellsgenerate one or more paracrine signals, and wherein the one or moreparacrine signals act on the administered cardiac stem cells andresident cardiac stem cells to increase the viability of theadministered cardiac stem cells and the resident cardiac stem cells,thereby reducing the incidence of apoptosis in the cardiac tissueaffected by the myocardial infarction.

In several embodiments the administration reduces the expression ofapoptotic markers on cardiac cells affected by the myocardialinfarction, thereby indicating a reduction in one or more portions ofthe apoptotic cascade. For example, in one embodiment, the decrease inapoptosis is associated with reduced Caspase 3 expression. In someembodiments, administration reduces the amount of plasma membrane damageon the cardiac cells affected by the myocardial infarction. In stilladditional embodiments, anti-apoptotic signals or markers are increased.For example, in one embodiment, the decrease in apoptosis is associatedwith an increase in Akt expression. As a result, in several embodiments,apoptosis is decreased by about 5% about 10%, about 15%, about 20%, ormore. In several embodiments, the decrease in apoptosis occurs withinseveral hours after administration of the cardiac stem cells. However,in several embodiments, the decrease in apoptosis occurs within severaldays after administration of the cardiac stem cells. In someembodiments, the decrease in apoptosis is associated with increasedcardiac function.

In some embodiments, damaged or diseased cardiac tissue is the result ofa reduced blood supply to a region of the cardiac tissue. For example, amyocardial infarction may result from the partial or total blockage of avessel providing oxygenated blood to one or more regions of the heart.In some embodiments, the major vessels may be affected, while in someembodiments, minor blockages (e.g., to the arterioles) may also damagethe cardiac tissue. Thus, in order to combat the deleterious effects ofreduced blood supply, there is provided a method for increasingangiogenesis in a heart having been affected by a myocardial infarction,comprising administering cardiac stem cells to a subject having a heartaffected by a myocardial infarction, wherein the administered populationof cardiac stem cells generate one or more paracrine signalspost-administration and prior to the destruction, and wherein the one ormore paracrine signals increase the level of angiogenesis in the cardiactissue affected by the myocardial infarction.

In several embodiments, at least a portion of the administeredpopulation of cardiac stem cells is destroyed by the subject's immunesystem. As a result, there may not be long-term survival of the entirepopulation of administered cells. However, as a result of the paracrinesignals, at least a portion of the beneficial effects of theadministered cells carries on beyond the time at which the cells aredestroyed. In some embodiments, the administered cells die on their owntime frame, and are simply removed from the tissue by endogenousmechanisms (e.g., phagocytosis). In some embodiments, the combination ofthe administered cells themselves (a direct repair mechanism) works inconcert with the induced paracrine cascade (either from the administeredcells or from the endogenous cells) to effect the increasedangiogenesis.

In several embodiments, the increased angiogenesis results in anincrease in vessel density in the cardiac tissue of the subject. In someembodiments, the vessel density is increased by about 2-fold, about3-fold, about 5-fold, or greater. In some embodiments, a 10% increase, a15% increase, a 20% increase, or greater is achieved. In severalembodiments, the increased angiogenesis increases the length of existingblood vessels. In some embodiments, the vessel length is increased byabout 2-fold, about 3-fold, about 5-fold, or greater. In someembodiments, a 10% increase, a 15% increase, a 20% increase, or greateris achieved. In several embodiments, combinations of increased vesseldensity length and increased density are achieved. In addition to theeffects on existing vessels, in several embodiments, the increasedangiogenesis increases the formation of new blood vessels. In someembodiments, a 5%, 10%, 15% or greater increase in new vessels isachieved. In combination with the positive effects on existing vessels,blood supply is increased to the region of damaged or diseased cardiactissue, which, in several embodiments, provides increased functionand/or viability to the region. In several embodiments, the increasedvessel density, increased vessel length, and/or the new vessels areassociated with improved function of about 5%, 10%, 15%, or greater inat least one of left ventricular percent fractional area and leftventricular ejection fraction.

In several embodiments, the paracrine signal comprises release of VEGFfrom the cardiac stem cells. In some embodiments, additionalpro-angiogenic factors are also generated by the cardiac stem cells,including endogenous cardiac stem cells.

In several embodiments, the plurality of cardiac stem cells is harvestedfrom healthy donor cardiac tissue. Depending on the amount of cellsdesired or required for administration, the plurality of the harvestedcardiac stem cells is expanded in culture to yield the population ofcardiac stem cells. While in several embodiments, the cardiac stem cellsare allogeneic with respect to the subject, in other embodiments, thecardiac stem cells are autologous with respect to the subject.

In addition to the administration of regenerative cells to a subject,there is also provided a method of regenerating cardiac tissue in anindividual having damaged cardiac tissue, comprising isolating apopulation of regenerative cells from cardiac tissue of a donor,expanding the population of regenerative cells in culture, wherein theregenerative cells in culture generate one or more paracrine factors,isolating the one or more paracrine factors from the culture, whereinthe isolated paracrine factors are suitable for administration to adamaged heart of an individual, and wherein, after administration, oneor more paracrine factors facilitate the formation of new cardiac tissuein the individual. In several embodiments, said damaged cardiac tissueis a result of myocardial infarction. In some embodiments,administration of said one or more paracrine factors results in adecrease in infarct size. In additional embodiments, the one or moreparacrine factors improves one or more of the viability or function ofthe damaged cardiac tissue. In one embodiment, administration of one ormore paracrine factors results in at least a 15% improvement in leftventricular function

In several embodiments, there is also provided a method of improving thecardiac function of an individual having damaged cardiac tissue,comprising identifying a subject having damaged cardiac tissue andadministering to said subject one or more paracrine factors, whereinsaid paracrine factors are obtained from a population of regenerativecells in culture, wherein said regenerative cells were isolated from thecardiac tissue of a donor, wherein said population of regenerative cellscomprises cardiac stem cells and, wherein, after administration, saidone or more paracrine factors induce formation of new functional cardiactissue and/or improve the function of the damaged cardiac tissue of saidsubject.

In several embodiments, said cardiac stem cells are selected from thegroup consisting of cardiospheres, cardiosphere-derived cells, and asubsequent generation of cardiospsheres. In one embodiment, said one ormore paracrine factors comprise one or more of VEGF, HGF, and IGFI.

Additionally, there is provided a method of increasing the function ofcardiac tissue in an individual having damaged cardiac tissue,comprising identifying a subject having damaged cardiac tissue,administering to said subject one or more paracrine factors selectedfrom the group consisting of VEGF, HGF, and IGFI, wherein said paracrinefactors are obtained from a population of regenerative cells in culture,wherein said regenerative cells were isolated from the cardiac tissue ofa donor, wherein said population of regenerative cells comprises cardiacstem cells, wherein said cardiac stem cells comprise one or more ofcardiospheres, cardiosphere-derived cells, and a subsequent generationof cardiospsheres, wherein, after administration, said one or moreparacrine factors recruit endogenous cardiac cells to the damagedcardiac tissue, wherein said recruited cells repair said damaged cardiactissue, thereby improving the function of the damaged cardiac tissue ofsaid subject. In several embodiments, the one or more paracrine factorsinduce production of paracrine factors by endogenous cells at or nearthe site of administration, thereby further increasing repair of saiddamaged cardiac tissue. In several embodiments, such method result in atleast a 15% improvement in left ventricular function.

In several embodiments, the donor is allogeneic with respect to theindividual, while in other embodiments, the donor and the recipientindividual are the same. Thus, there are provided herein both cell-basedand cell-free methods of generating and/or repairing cardiac tissue.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1I depict specimen processing for cardiosphere growth andCardiosphere-Derived Cell (CDC) expansion. FIG. 1A depicts a schematicof the steps involved in certain embodiments of specimen processing.FIG. 1B depicts a human endomyocardial biopsy fragment on day 1. FIG. 1Cdepicts a human explant 3 days after plating. FIG. 1D depicts the edgeof a human explant 13 days after plating and showing stromal-like andphase-bright cells. FIG. 1E depicts results of sub-population selectionperformed using cardiosphere-forming cells. c-kit⁺ cells were 90.0±4.7%CD105⁺, and c-kit⁻ cells were 94.0±0.8% CD105⁺ (n=3). FIG. 1F depictshuman cardiospheres on day 25, 12 days after collection ofcardiosphere-forming cells. FIG. 1G depicts human CDCs during passage 2,plated on fibronectin for expansion. FIG. 1H depicts cumulative growthcurves for 11 specimens from untransplanted patients over the course of4 months. FIG. 1I depicts growth for 59 specimens from transplantedpatients. Day 0 corresponds to the date the specimen was collected andcell number on that day is plotted as 1 on the log scale, since nocardiosphere-forming cells had yet been harvested from the specimen.

FIGS. 2A-2C depict cardiosphere and CDC phenotypes. FIG. 2A depictsexpression of c-kit throughout the core of a cardiosphere and CD105 onthe periphery. FIG. 2B depicts expression of cardiac MHC and TnIprimarily on cardiosphere periphery. FIG. 2C depicts c-kit and CD105expression levels in CDCs at passage 2 shown for one representativespecimen (n=3 and n=2).

FIGS. 3A-3E depict engraftment and regeneration. Engraftment of CDCs(FIG. 3A) or fibroblasts (FIG. 3B) is depicted 20 days after injectionin heart sections double stained for H&E and beta-galactosidase.Infiltration of CDCs is seen as a distinct band, while a rare group of afew fibroblasts can be detected in some sections. Masson's trichromestaining as used to calculate myocardial regeneration is shown for arepresentative CDC-injected mouse (FIG. 3C) and fibroblast-injectedmouse (FIG. 3D). FIG. 3E depicts the percent of viable myocardium foundwithin the infarcted area in CDC (n=8), PBS (n=4), andfibroblast-injected (n=4) groups (* p<0.01).

FIGS. 4A-4F depicts functional cardiac improvement. Long-axis views froman echocardiogram performed after 20 days in a CDC-injected mouse areshown. FIG. 4A depicts end-diastole while FIG. 4B depicts end-systole.Yellow lines trace around the left ventricular area used for thecalculation of LVEF and LVFA. (FIG. 4C and FIG. 4D). FIG. 4E shows theleft ventricular ejection fractions (LVEF) for the three experimentalgroups after 20 days (CDC n=8, PBS n=7, Fibroblast n=4; *p<0.01). LVEFwas calculated as

100×(LVVolume_(diastole)−LVVolume_(systole))/LVVolume_(diastole)

and left ventricular volume (LVVolume) was calculated from long-axisviews assuming a prolate ellipsoid. FIG. 4F depicts left ventricularpercent fractional area (LVFA) for the three experimental groups after20 days. *p<0.01. LVFA was calculated as:

100×(LVArea_(diastole)−LVArea_(systole))/LVArea_(diastole)

FIGS. 5A-5C depict quantification of cardiac tissue regeneration. FIG.5A shows Masson's trichrome staining for a representative CDC-injectedmouse. The total infarct zone is outlined in yellow in FIG. 5B and FIG.5C. FIG. 5B depicts areas of fibrosis in red after image processing.FIG. 5C depicts areas of viable myocardium in red after imageprocessing. Six sections were analyzed per animal and an average taken.

FIGS. 6A-6F depict an engraftment time course. FIG. 6A depicts the bolusof injected cells on day 0 in an H&E stained section of cardiac tissue.FIG. 6B depicts engraftment of CDCs 8 days after injection. FIG. 6C andFIG. 6D depict engraftment of CDCs 20 days after injection. FIG. 6E andFIG. 6F depict corresponding higher magnification views of FIG. 6C andFIG. 6D demonstrating colocalization of lac-Z-positive CDCs and viablemyocardium.

FIGS. 7A-7C depict the growth of CDCs generated from 2 whole hearts andsubjected to various growth conditions. FIG. 7A depicts CDC yield fromsamples taken from different regions of the heart. FIG. 7B depicts theeffect of tissue storage in cold cardioplegia for up to 6 days orfreezing and thawing. FIG. 7C depicts a comparison of yield of CDCs fromeach of the sample hearts grown under the same conditions.

FIGS. 8A-8B depict phenotypic identity of human CDCs. FIG. 8A depictssurface immunophenotype by flow cytometry of human CDCs from multiplepatients. Dual-labeled analysis depicting mesenchymal and progenitor CDCsub-populations is shown in FIG. 8B.

FIGS. 9A-9C depict characteristics of differentiating CDCs. FIG. 9Adepicts sarcomeric organization in human CDCs co-cultured with ratneonatal ventricular myocytes. FIG. 9B depicts inwardly rectifyingpotassium current recorded form CDCs, which is consistent withcardiomyocyte ventricular phenotype. FIG. 9C depicts L-type calciumcurrent I_(Ca,L) recorded from CDCs transduced with the beta subunit ofthe L-type calcium channel, indicating the presence of the pore-formingalpha subunit.

FIGS. 10A-10B depict CDCs forming tube matrices in an angiogenesisassay. Human CDCs (FIG. 10A) form tube-like networks at early timepointsand undergo complex morphological changes at late timepoints. This is incomparison to human umbilical vein endothelial cells (HUVECs) (FIG. 10B)which form distinct tube networks.

FIGS. 11A-11B depict engraftment of human CDCS. FIG. 11A depictsluciferase-labeled cells detected in the heart at 1 day, 4 days, and 1week post-administration. FIG. 11B depicts peak signals detected foreach of 3 mice.

FIGS. 12A-12K depict detection of human CDCs in injected mouse hearts.Mouse hearts excised at different timepoints following MI and deliveryof human CDCs are shown in FIGS. 12A-12C. Human nuclei are identified ateach timepoint by green fluorescence overlaid on the blue fluorescenceof all nuclei (FIGS. 12D-12K). INF=infarct. BZ=border zone.

FIG. 13 depicts a summary of echocardiography on mice injected withCDCs, normal human derived fibroblasts, or phosphate buffered saline.CDC-injected animals maintained function after MI, while NHDF- andPBS-injected declined.

FIG. 14 depicts the effect of various CDC subpopulations on murinecardiac function. CDC-injected animals outperformed animals injectedwith either c-kit or CD90 sub-populations.

FIGS. 15A-15R depict differentiation of human CDCs at varioustime-points in infarcted mice.

FIGS. 16A-16P depict formation of cardiomyocytes and non-cardiomyocytesfrom human CDCs at 6 weeks post-MI. Human nuclei of interest areoutlined in FIGS. 16E-16H. Example cardiomyocyte nuclei are shown inFIGS. 16M-16P at higher magnification.

FIG. 17 depicts a schematic of in vitro and in vivo experimental designsto evaluate functional effects of paracrine signals released fromregenerative cells.

FIGS. 18A-18C depict protein array analysis of serum free conditionedmedia collected from the culture of regenerative cells, specificallycardiospheres and CDCs.

FIGS. 19A-19E depict in vitro analysis of VEGF, HGF and IGFI secretion.

FIGS. 20A-20F depict the gene expression profiles of various growthfactor receptors on cardiospheres and CDCs.

FIGS. 21A-21K depict the effects of conditioned media from regenerativecells on cultured cells. FIGS. 21A-21E depict the pro-survival effectsof conditioned media from regenerative cells on neonatal rat ventricularmyocytes (NVRMs) and pro-angiogenic (FIGS. 21F-21K) effects ofconditioned media from regenerative cells on cultured HUVEC cells.

FIG. 22 depicts results of growth factor and cytokine protein expressionanalysis in infarcted regenerative cell-injected mouse hearts.

FIGS. 23A-23D depict assessment of tissue viability and cardiac tissueperfusion after administration of regenerative cells.

FIGS. 24A-24D depict an evaluation of the amount of regenerative cellcontribution to capillaries and muscle tissue formed in the border zoneof an induced infarction.

FIGS. 25A-25C depict an analysis of infarct size evaluated by Masson'strichrome staining.

FIGS. 26A-26H depict MHC Class I and II expression for human CDCs beforeand after interferon stimulation.

FIGS. 26I-26J depict the fractional change in lymphocyte proliferationnormalized to syngeneic co-culture for allogeneic and xenogeneicco-cultures.

FIGS. 26K-26M depict the associated lymphocyte infiltration insyngeneic, allogeneic, and xenogeneic co-cultures.

FIG. 26N depicts the index of lymphocyte proliferation induced byvarious co-cultures.

FIG. 26O depicts data related to the quantification of inflammatorycytokines after co-culture.

FIG. 27A depicts a graphical experimental scheme employed to studyengraftment and function of CDCs.

FIG. 27B depicts the experimental and control groups used to evaluateengraftment and function.

FIG. 27C depicts data related to 1-week cell engraftment in varioustransplant groups.

FIG. 27D depicts data related to 3-week cell engraftment in varioustransplant groups.

FIGS. 28A-28C depict hematoxylin and eosin staining of syngeneic,allogeneic, or xenogeneic treated cardiac tissue. Analysis revealedsignificant evidence of an immune reaction in xenogeneic (28A) heartsections, but very little in syngeneic (28B) or allogeneic (28C)sections. Example images are shown at 3 weeks post-MI.

FIGS. 28D-28G depict 1-week and 3-week data related to the rejectionscore for various transplant types at various regions throughout theinfarct scar or border zone.

FIGS. 28H-28J depict immunohistochemistry data related to cellinfiltration post-transplant.

FIGS. 28K ₁-28K₁₅ depict immunohistochemistry further defining the typesof infiltrating lymphocytes in each transplant.

FIGS. 28L-28M depict data related to monocyte infiltration in eachtransplant type at 1 and 3 weeks.

FIGS. 29A-29E represent data and analysis of T-cell, B-cell, andmacrophage infiltration in various treatment groups at 1 and 3-weekspost-MI. Engraftment of GFP-labeled CDCs and the level of T cell (CD3⁺,CD4⁺, or CD8⁺), B cell (CD45R⁺), or macrophage (CD68⁺) infiltrationsurrounding the cells 3 weeks post-MI is shown for animals who receivedsyngeneic (29A), allogeneic (29B), or xenogeneic (29C) CDCs. The numberof cells per high power field is quantified 1 week (29D) and 3 weekspost-MI (29E).

FIGS. 30A-30G depict quantification of serum concentrations ofpro-inflammatory cytokines IFN-γ, IL-113, KC/GRO, TNF-α, (FIGS. 30A,30B, 30F, and 30G, respectively), or the anti-inflammatory cytokinesIL-13, IL-4, and IL-5 (FIGS. 30C, 30D, and 30E, respectively).

FIG. 31A depicts morphometric analysis of explanted hearts 3 weeks postinfarction in syngeneic (upper left), allogeneic (upper right),xenogeneic (lower left) and control (lower right) groups.

FIG. 31B depicts infarct size in the various transplant groups whileFIG. 31C depicts infarcted wall thickness.

FIGS. 31D-31G depict fractional area change (31D), ejection fraction(31E), fractional shortening (31F), and treatment effect (31G) for thevarious transplant groups.

FIGS. 31H-31V depict immunohistochemistry related to the location oftransplanted cells as well as markers for cell cycle, stem cell markers,and endothelial cell markers.

FIGS. 31W-31Z depict data at 1 and 3 weeks for, respectively,Ki67/smooth muscle actin, BrdU expression/smooth muscle actinexpression, cKit expression, and vessel density.

FIG. 31AA depicts protein analysis of growth factor secretion at varioustime points post-MI.

FIGS. 31BB-31DD depict data for VEGF (31BB), IGF (31CC), and HGF (31DD)in syngeneic, allogeneic, or control groups.

FIGS. 32A-32B depict schematics of two study designs disclosed herein.

FIG. 33 depicts the percentage of CDCs retained, after injecting intothree different areas of pig heart, as determined by an in vitroluciferase assay 24 hours after injection.

FIGS. 34A-34C depict changes in LV function as measured byventriculography. Paired analysis of LVEF before injection and 8 weekslater in controls and CDC-injected pigs is shown (34A). Average LVEF forpigs in the CDC-injected group was significantly higher than those inthe control group 8 weeks after injection (34B). Treatment effects inthe two groups as absolute change in LVEF (34C).

FIGS. 35A-35C depict various echocardiographic measurements. FIG. 35Ashows that Emax in the cardiosphere group was significantly higher thanin placebo-injected pigs. Emax in the CDC group was not significantlyhigher than placebo-treated animals. (Levene's test p<0.05,Kruskal-Wallis comparison p=0.003. Placebo vs. CDC, p=NS; Placebo vs.cardiosphere, p=0.03). Representative families of PV loops are shownbelow the graph for placebo, CDC and cardiosphere-treated animals. FIG.35B shows the change in diastolic volume (Final EDV-Baseline EDV) issignificantly lower in the cardiosphere treated group than the CDCtreated group, with a trend to being lower than the placebo group.(ANOVA p=0.02). FIG. 35C shows that the changes in end-diastolicpressure measurements demonstrated a significantly higher fall inend-diastolic pressure in cardiosphere-injected animals compared to CDCtreated animal (ANOVA p<0.01; cardiospheres vs. CDCs p=0.001). Thisindicates that, in some embodiments, the ventricles ofcardiosphere-injected hearts are more responsive to treatment afterinfarction CDC-treated hearts. However, in some embodiments, CDC-treatedhearts are responsive to an equal or greater degree.

FIGS. 36A-36C depict improved regional contractility incardiosphere-injected pigs relative to sham-injected controls.

FIGS. 37A-37B depict two examples of islands of cardiomyocytes withlacZ-positive nuclei in the periinfarct zone, one from each animal thatreceived intramyocardial genetically-labeled CDCs.

FIGS. 38A-38F depict schematics of various cell processing and cellbanking procedures disclosed herein.

FIGS. 39A-39B depict correlation of LVEF values measured independentlyby two blinded, experienced echocardiographers. Measurements for eachanimal from the two readers show good correlation at baseline (39A) and3 weeks after treatment (39B).

FIGS. 40A-40E depict characteristics of the various stem cell typesevaluated. FIGS. 40A-40D depict phase-bright images of CDCs (shown in40A), bone marrow-derived mesenchymal stem cells (BM-MSC, shown in 40B),adipose tissue-derived mesenchymal stem cells (AD-MSC, shown in 40C),and bone marrow mononuclear cells (BM-MNC, shown in 40D) in culture.FIG. 40E displays the expression of certain surface markers on each celltype.

FIGS. 41A-41J depict the secretion of a variety of paracrine factors byvarious stem cell types. FIGS. 41A-41F display the concentrations ofeach factor by each cell type. FIGS. 41G-41K display a relative paracinefactor profile for each cell type.

FIGS. 42A-42C depict the in vitro production of growth factors from ratcells. The concentrations of VEGF (42A), IGF-1 (42B), and HGF (42C)measured by ELISA are shown.

FIGS. 43A-43D depict analysis of in vitro myogenic differentiation andangiogenesis assay. FIG. 43A shows that Troponin T, with distinctmyocyte-like appearance, was expressed spontaneously in a fraction ofCDCs cultured for 7 days. This cardiac-specific marker was rarelyexpressed in BM-MSCs, AD-MSCs, and BM-MNCs. FIG. 43B depictsquantitative analysis of Troponin T expression in CDCs (9% of the cellspositive), BM-MSCs (0.4% positive) and AD-MSCs and BM-MNCs(approximately 0.1% positive). FIG. 43C depicts CDCs, BM-MSCs, andAD-MSC-derived production of capillary-like tube formations inextracellular matrix. BM-MNCs did not form similar structures underthese conditions. FIG. 43D depicts quantitation and comparison of tubeformation capacity by the different cell types. Bars=50 um.

FIGS. 44A-44B depict the in vitro resistance of different types of humancells to oxidative stress. FIG. 44A shows representative images ofTUNEL-positive cells (red) after 24 hours exposure to 100 mM H2O2. FIG.44B depicts quantitative assessment of apoptotic cells. The number ofTUNEL-positive cells was lower in CDC group compared to BM-MNC groupwith 100 mM H2O2. Bar=50 mm.

FIGS. 45A-45B depict in vitro resistance of different cell typesisolated from the same rat, to oxidative stress. FIG. 45A showsrepresentative images of TUNEL-positive cells (red) after 24 hours'exposure to 100 mM H2O2. FIG. 45B shows quantitative analysis showedthat the number of apoptotic cells was significantly lower in CDCs,BM-MSCs, and AD-MSCs than in BM-MNCs with 100 mM H₂. Bar=50 mm.

FIGS. 46A-46B depict in vitro myogenic differentiation of different celltypes (all isolated from the same rat). FIG. 46A shows a fraction of theCDCs positively expressed the cardiac specific marker Troponin T, withdistinct myocyte-like appearance. Troponin T expression was rarelyobserved in BM-MSCs, AD-MSCs, and BM-MNCs. FIG. 46A depicts thequantitative assessment of Troponin T expression in different cell typesis shown. Bar=50 mm.

FIGS. 47A-47C depict cell engraftment and in vivo myogenicdifferentiation. FIG. 47A Immunostaining shows some human CDCs (green,HNA) expressing α-sarcomeric actin, indicating myogenic differentiation,3 weeks after implantation into infarcted mice hearts. FIG. 47BQuantitation of engraftment (HNA+ cells). C) Quantitation ofcardiomyocytes differentiated from transplanted human cells (HNA+/αSA+cells). Bar=20 μm.

FIGS. 48A-48C depict results of cell apoptosis studies. FIGS. 48A and48B are representative images of TUNEL-positive cells in the infarctedhearts of mice 3 weeks after cell treatment with CDCs (48A) and PBS(48B). FIG. 48C depicts quantitative assessment of TUNEL-positive cellsin the myocardium of mice treated with different cell types and control,is shown. Bar=500 μm.

FIG. 49 depicts cardiac function of mice treated with the various celltypes. LVEF at baseline (4 hrs post-MI) did not differ among groups,indicating a similar infarct size in animals of all groups. After 3weeks, LVEF was higher in mice implanted with CDCs, compared to animalstreated with cells of non-cardiac origin. Implantation of BM-MSCs alsoimproved cardiac function, compared to controls injected with salineonly. Data are presented as mean±SEM.

FIGS. 50A-50H depict Ventricular remodeling after treatment with variouscell types. FIGS. 50A-50F are representative images of Masson's stainingof infarcted mice hearts, after implantation of different types of humancells or saline injection only. Quantitative analyses of LV wallthickness (50G) and infarct perimeter (50H) show that remodeling wasattenuated more efficiently by CDC implantation, compared with BM-MSCs,AD-MSCs, and BM-MNC treatment, although implantation of BM-MSCs,AD-MSCs, and BM-MNCs resulted in less remodeling compared to controltreatment with saline injection only.

FIGS. 51A-51E depict a comparison of purified c-kit⁺ stem cells andunsorted CDCs. FIG. 51A shows LVEF 3 weeks after infarction. LVEF washigher in mice that received unsorted CDCs than those with c-kit⁺ cellspurified from the same CDCs. FIGS. 51B-51E show that although the samenumber of cells was used for culture, the purified c-kit⁺ stem cellsreleased less VEGF, SDF, IGF-1, and HGF than the unsorted CDCs.

DETAILED DESCRIPTION

Cell therapy, the introduction of new cells into a tissue in order totreat a disease, represents a promising new method for repairing orreplacing diseased tissue with healthy tissue. Therefore, in severalembodiments described herein, methods of isolating, culturing,preparing, and introducing regenerative cells into a recipient areprovided and result in one or more of treatment of symptoms of a cardiacdisease, improvement in cardiac function, and/or regeneration of cardiactissue in the recipient. In several embodiments, the cardiac disease isthe result of one or more of an acute heart failure or chronic heartfailure. In some embodiments, the disease creates damaged to the cardiactissue due to one or more of ischemia, reperfusion, or infarction.

As used herein, the term “regenerative cells” shall be given itsordinary meaning and shall include mixed cell populations (e.g.,cardiospheres) and derivatives thereof (e.g., CDCs and secondarygenerations of cardiospheres (IICSps)), unless explicitly indicatedotherwise. Regenerative cells include cells that directly repair tissue(e.g., stem cells) and cells that promote tissue repair (e.g., throughparacrine effects or other signaling events).

General

Several embodiments disclosed herein provide methods for expandingpopulations of resident stem cells from organs, such that only smallinitial samples are required. Such small initial samples can be obtainedrelatively non-invasively, for example, by a simple percutaneous entry.Such samples can be obtained using a percutaneous bioptome, in someembodiments. The bioptome can be used to access a tissue sample from anyorgan source, including heart, kidney, liver, spleen, and pancreas.Particularly suitable locations within the heart which can be accessedusing a bioptome include, but are not limited to, the crista terminalis,the right ventricular endocardium, the septal or ventricle wall, and theatrial appendages. These locations have been found to provide abundantstem or progenitor cells. Accessing such locations is facilitated by useof a bioptome which is more flexible than the standard bioptome used foraccessing the right ventricular endocardium for diagnostic purposes.Preferably the bioptome is also steerable by an external controller.Such procedures are enable collection of tissue on an out-patient basiswithout major surgery or general anesthesia. While percutaneous biopsyis employed in some embodiments, in other embodiments, severalembodiments employ explanted tissues (e.g., those removed from a subjectand under evaluation for transplantation).

One of the advantages of several embodiments disclosed herein thatenables use of a small biopsy sample (or small non-biopsy samples) as astarting material is the collection of a cell population which haspreviously been ignored or discarded. In one embodiment, this cellpopulation is formed by treating the sample with a protease andharvesting or collecting the cells that are liberated from the sample.The use of these liberated cells enhances the rate of cell populationexpansion. Non-limiting examples of proteases which can be employedinclude collagenase, matrix metalloproteases, trypsin, and chymotrypsin.This technique can be applied to any organ from which resident stemcells are desired, including, for example, heart, kidney, lung, spleen,pancreas, and liver. In some embodiments, the mass of tissue collectedto isolated resident stem cells is roughly equivalent, regardless of themanner in which the tissue was obtained. For example, in someembodiments, the amount of tissue collected ranges from about 10 mg oftissue to about 1000 mg of tissue. For example, in some embodiments, theamount of tissue collected ranges from about 20 mg of tissue to about500 mg of tissue. In some embodiments, the amount of tissue collectedranges from about 10-50 mg of tissue, about 50-100 mg of tissue, about100-150 mg of tissue, about 150-200 mg of tissue, about 200-250 mg oftissue, about 250-300 mg of tissue, about 300-350 mg of tissue, about350-400 mg of tissue, about 400-450 mg of tissue, about 450-500 mg oftissue, and overlapping ranges thereof. In some embodiments, a total ofno more than 20 mg of tissue is collected. In some embodiments, a totalof no more than 30 mg of tissue is collected. In some embodiments, atotal of no more than 40 mg of tissue is collected. In some embodiments,a total of no more than 50 mg of tissue is collected. In someembodiments, a total of no more than 60 mg of tissue is collected. Insome embodiments, a total of no more than 70 mg of tissue is collected.In some embodiments, a total of no more than 80 mg of tissue iscollected. In some embodiments, a total of no more than 90 mg of tissueis collected. In some embodiments, a total of no more than 100 mg oftissue is collected. In several embodiments, the mass of tissuecollected from any single biopsy or collection ranges between about10-20 mg, including about 11, 12, 13, 14, 15, 16, 17, 18, and 19 mg. Inseveral embodiments, the mass of tissue collected from any single biopsyor collection ranges between about 20-30 mg, including about 21, 22, 23,24, 25, 26, 27, 28, and 29 mg. In several embodiments, the mass oftissue collected from any single biopsy or collection ranges betweenabout 30-40 mg, including about 31, 32, 33, 34, 35, 36, 37, 38, and 39mg. In several embodiments, the mass of tissue collected from any singlebiopsy or collection ranges between about 40-40 mg, including about 41,42, 43, 44, 45, 46, 47, 48, and 49 mg.

Resident stem cells are those which are found in a particular organ. Asdiscussed below in more detail, it is believed that the stem cells foundin a particular organ are not necessarily pluripotent, but rather, arecommitted to a particular branch of differentiation. Thus in the heart,one expects to find cardiac stem cells, and in the kidney one expects tofind kidney stem cells. Despite this, in several embodiments some of thestem cells isolation and expanded using the methods disclosed herein areable to develop into cells of an organ other than the one from whichthey were obtained.

Cardiospheres are self-associating aggregates of cells which have beenshown to display certain properties of cardiomyocytes. Cardiosphereshave been shown to “beat” in vitro. They are excitable cells andcontract in synchrony. In one embodiment, the cells which form thecardiospheres have been obtained from heart biopsies. In one embodiment,the cells which form the cardiospheres have not been obtained from heartbiopsies, but rather have been obtained from a whole heart, or a portionthereof. The cardiospheres can be disaggregated using standard meansknown in the art for separating cell clumps or aggregates, including,but not limited to trituration, agitation, shaking, blending. In oneembodiment, cardiospheres are disaggregated to single cells. In severalembodiments they are disaggregated to smaller aggregates of cells. Inseveral embodiments, after disaggregation, the resultant cells are grownon a solid surface, such as a culture dish, a vessel wall or bottom, amicrotiter dish, a bead, flask, roller bottle, etc. The surface can beglass or plastic, for example. In one embodiment, the cells are capableof adhering to the material of the solid surface. The solid surface isoptionally coated with a substance which encourages adherence in someembodiments. Several such substances are known in the art and include,without limitation, fibronectin. hydrogels, polymers, laminin, serum,collagen, gelatin, and poly-L-lysine. In several embodiments, growth onthe surface is monolayer growth. These cells are cardiosphere-derivedcells (CDCs).

In several embodiments, after growth of CDCs, they are directlyadministered to a mammal in need thereof. In one embodiment the CDCs areoptionally grown under conditions which favor formation ofcardiospheres. In one embodiment, the cells themselves are notadministered, but one or more substances contacted with or released fromthe cells are administered. Repeated cycling between surface growth andsuspension growth (cardiospheres) leads to a rapid and exponentialexpansion of desired cells. In one embodiment the cardiosphere phase iseliminated and CDCs are repeatedly expanded through growth on a surfacewithout forming cardiospheres at each passage.

Several embodiments, of the culturing processes disclosed herein(whether culturing CDCs on surfaces or cardiospheres in suspension) areperformed in the absence of exogenous growth factors. In one embodiment,fetal bovine serum is used, but other factors are viewed as expendable.For example the cells of the present invention are readily cultured inthe absence of added EGF, bFGF, cardiotrophin-1, and thrombin.

Mammals which can be the donors and recipients of cells are not limited.Thus, while in several embodiments, humans provide both the cells andare the recipients, often other mammals will be useful. Pig cells can betransplanted into humans, for example. Such cross-speciestransplantation is known as xenogeneic transplantation. Thetransplantation can also be allogeneic, syngeneic, or autologous, allwithin a single species. Suitable mammals for use in such transplantsinclude pets, such as dogs, cats, rabbits; agricultural animals, such ashorses, cows, sheep, goats, pigs, as well as humans.

Administration of cells to a mammal can be by any means known in theart. Cardiac cells can be delivered systemically or locally to theheart. In several embodiments, the administered cells are not in theform of cardiospheres, but rather are CDCs. In some embodiments,cardiospheres are delivered. As discussed above, they have the capacityto form cardiospheres under suitable conditions. Local administrationcan be by catheter or direct (e.g., during surgery). Systemicadministration can be by intravenous or intraarterial injections,perfusion, or infusion. In those embodiments, employed systemicadministration, the administered cells migrate to the appropriate organ,e.g., the heart, if the cells are derived from resident heart stemcells.

The beneficial effects which are observed upon administration of thecells to a mammal may be due to the cells per se in some embodiments.For example, in some embodiments the engraftment of cells produces afavorable outcome. However, in some embodiments, the beneficial effectsare due to products which are expressed by the cells (e.g., a surfacemarker that interacts with host tissue) or secreted, released orotherwise delivered to the recipient tissue by the cells. In severalembodiments cytokines or chemokines or other diffusible factors,including but not limited to paracrine factors (e.g., growth factors)stimulate resident cells to grow, reproduce, or perform better. In someembodiments, the combination of the cells and diffusible factors providethe beneficial effects.

As discussed below, in some embodiments, an effective dose of cardiacstem cells will typically be between 1×10⁶ and 100×10⁶. In someembodiments, the dose is between 10×10⁶ and 50×10⁶. Depending on thesize of the damaged region of the heart, more or less cells may be used.For example, when treating a larger region of damage, a larger dose ofcells may be used, and a small region of damage may require a smallerdoes of cells. On the basis of body weight of the recipient, aneffective dose may be between 1 and 10×10⁶ per kg of body weight,preferably between 1×10⁶ and 5×10⁶ cells per kg of body weight. Patientage, general condition, and immunological status may be used as factorsin determining the dose administered.

Diseases which can be treated using the methods and compositionsdisclosed herein include acute and chronic heart disease. For example,hearts having been subjected to an ischemic incident, subjected ofchronic ischemia, or congestive heart disease are treated in someembodiments. In some embodiments, patients are candidates for hearttransplants or recipients of heart transplants. In additionalembodiments, hearts which are damaged due to trauma, such as damageinduced during surgery or other accidental damage, are treated accordingto the methods disclosed herein.

The cell populations which are collected, expanded, and/or administeredaccording to the present invention can optionally be geneticallymodified, according to several embodiments. In one embodiment, they aretransfected with a coding sequence for a protein, for example. Theprotein can be beneficial for diseased organs, such as hearts.Non-limiting examples of coding sequences which can be used includewithout limitation akt, connexin 43, other connexins, HIF1-alpha, VEGF,FGF, PDGF, IGF, SCF, myocardin, cardiotrophin, L-type calcium channelalpha-subunit, L-type calcium channel beta-subunit, and Nkx2.5. Thecells may be conveniently genetically modified before the cells areadministered to a mammal. Techniques for genetically modifying cells toexpress known proteins are well known in the art. As discussed herein,in several embodiments, genetic modification is not necessary as thebeneficial effects which are observed upon administration of the cellsto a mammal are due to the engraftment of the administered cells, due toproducts which are expressed by the cells (e.g., a surface marker thatinteracts with host tissue) or secreted, released or otherwise deliveredto the recipient tissue by the cells (e.g., cytokines or chemokines orother diffusible factors, such as paracrine factors) or combinationsthereof.

As discussed below, CDCs were easily harvested and readily expanded frombiopsy specimens as well as from transplant-ready hearts, and afteradministration regenerated myocardium and improve function in severalacute MI models. 69 of 70 patients had biopsy specimens that yieldedcells by the methods disclosed herein, making the goal of autologouscellular cardiomyoplasty attainable. In some embodiments, autologouscells are used, as the cells are a perfect genetic match and thuspresent fewer potential safety concerns than allogeneic cells. However,practical limitations with the use of autologous cells may arise fromthe delay from tissue harvesting to cell transplantation. To avoid thedelay, cell banks can be created of cardiac stem cells from patientswith defined immunological features. These should permit matching ofimmunological antigens of donor cells and recipients for use inallogeneic transplantation. Antigens for matching are known in the artof transplantation. Likewise, as disclosed below, in several embodimentsallogeneic cells need not be matched, as they present limited immuneresponses when administered. In some embodiments, this is due a shortresidence time of the cells themselves. In some embodiments, the choiceof cardiospheres or CDCs determines the residence time. In severalembodiments, CDCs reside in host tissues for about 3-6 weeks, and thenare destroyed by host mechanisms. However, despite the loss of some orall of the originally administered cells, beneficial effects are stillobserved, likely due to paracrine effects set in motion by the cells. Inseveral embodiments, the relatively short residence time of theadministered cells limits the immune response.

Previous clinical studies in which bone marrow-derived stem cells wereinjected into patients within 2 weeks following acute MI, resulted insignificantly improved LVEF with intracoronary infusion of 5-80×10⁶cells. As such, several million CDCs may constitute an effectivetherapeutic dose, in certain embodiments. From single bioptome ornon-biopsy specimens, millions of CDCs can be derived after just twopassages; if biopsies or sample collection were performed specificallyfor therapeutic purposes, the amount of starting material could easilybe scaled upwards by ten-fold or more, further improving the overallcell yield. In several embodiments, however, other variables, such asculture conditions, can increase the yield while allowing reduction inthe amount of starting material required.

In some embodiments, minimizing the number of passages for expansionwill minimize the risk of cancerous transformation of CDCs, a problemwhich has been observed in mesenchymal stem cells, typically after about6 or more passages. Another prominent risk of cell transplantation liesin the potential for arrhythmogenicity. Arrhythmias have not beendocumented with cardiac stem cells. Teratoma formation is also aconcern, though in several embodiments, teratoma formation is reduced ornon-existent. In one embodiment, teratoma formation can occur when cellsare not committed to forming a specific type of tissue.

In several embodiments, CDCs are derived from human biopsies (ornon-biopsy samples) without antigenic selection (including but notlimited to c-kit). In several embodiments, all cells that are shed fromthe initial heart specimen and which go on to contribute to theformation of cardiospheres. Thus, in several embodiments, cellsaccording to several embodiments of the invention differ fundamentallyfrom cardiac “stem cells” which have been isolated by antigenic panningfor one or another putative stem cell marker, for example c-kit.Nevertheless, CDCs include a sizable population of cells that exhibitstem cell markers, and the observed regenerative ability in vivo furthersupports the notion that CDCs include a number of resident stem cells.In some embodiments, a subfraction of CDCs suffices to produce thebeneficial effects; however, subfractionation may delay transplantationand raise regulatory concerns by introducing an artificial selectionstep. Thus, in several embodiments CDC or cardiosphere populations thathave not been enriched for any one marker (including but not limited toc-kit) are particularly advantageous, as the required manipulation andhandling of the cells is reduced and the resultant beneficial effectsare equivalent.

Adult human cardiac stem cells have been shown to respond to a limiteddegree to a state of cardiac hypertrophy by proliferation and myocardialregeneration and to acute ischemia by mobilization to the injury borderzone and subsequent regeneration, but often ultimately succumb toapoptosis in a chronic ischemic setting. Significant progress iscurrently being made identifying means of enhancing in vivo survival,mobilization, proliferation, and subsequent differentiation of CSCsusing animal models. The methods disclosed herein for ex vivo expansionof resident stem cells for subsequent autologous or allogeneictransplantation may give these cell populations, the resident and theexpanded, the combined ability to mediate myocardial regeneration to anappreciable degree. If so, cardiac stem cell therapy may well change thefundamental approach to the treatment of disorders of cardiacdysfunction.

Transplant and Regenerative Cell Types

With respect to the recipient of administered regenerative cells,several embodiments use allogeneic regenerative cells. In such atransplant, the donor and recipient are different individuals within thesame species. For example, a first mammal is the donor of the cells anda second mammal is the recipient of the cells. In several embodiments,the mammals are humans. In several embodiments, autologous stem cells(donor and recipient are the same) are used. In such embodiments, adonor's own regenerative cells are re-administered to the sameindividual. In several other embodiments xenogeneic (donor and recipientare different individuals from different species) regenerative cells areused. In some such embodiments, closely related phylogenetic species areused, such as humans and chimpanzees. In other embodiments, individualsfrom more distantly related species may be the donor and recipient, butimmunological compatibility is still possible. In several embodiments,syngeneic (genetically identical and thus, immunologically compatible)regenerative cells are used. Although adult regenerative cells areprovided in several embodiments of the invention, embryonic regenerativecells are used in one embodiment. However, many embodiments of theinvention obviate the need for embryonic stems cells. In someembodiments, other cell types are used, for example, myoblasts orperipheral blood-derived endothelial progenitors may be used.Additionally, induced pluripotent stem cells are used in someembodiments.

The Major Histocompatibility Complex (MHC) is large, gene-dense regionof the mammalian genome, which plays and important role in immune systemfunction. The proteins encoded by the MHC are expressed on the surfaceof cells and thus are involved in antigen presentation as well aslymphocyte recognition. MHC molecules effectively control the initiationof an immune response through identification of cells as “self” or“non-self.” Thus, MHC molecules are key targets in transplantationrejection.

The most well-known genes in the MHC region are the subset that encodeantigen-presenting proteins on the cell surface. In humans, these genesare referred to as human leukocyte antigen (HLA) genes. In humans, theMHC is divided into three regions: Class I, II, and III. HLA class Iantigens (A, B, and C) present peptides from inside the cell (includingviral peptides if present). HLA class II antigens (DP, DM, DOA, DOB, DQ,and DR) present antigens from outside of the cell to T-lymphocytes. HLAclass III antigens encode components of the complement system.

While class I and class II MHC molecules are structurally similar andboth present antigens to T-cells, their functions in the immune responsecascade are different. Class I molecules are found on virtually everycell in the human body while Class II molecules, in contrast, are onlyfound on B-cells, macrophages and other antigen-presenting cells. ClassI molecules present antigens to cytotoxic T-cells (CTLs) while class IImolecules present antigen to helper T-cells. This specificity of antigenpresentation leads into another difference, the type of antigenpresented. Class I molecules present endogenous antigens while class IImolecules present exogenous antigens. For example, an endogenous antigencould be a viral protein fragment or tumor protein. These endogenousantigens indicate internal cellular alterations that need to becontrolled so that they don't spread throughout the body. In contrast,exogenous antigens may comprise fragments of bacterial cells or viruses,e.g., non-self antigens, which are engulfed and processed by, forexample, a macrophage, and then presented to helper T-cells. The helperT-cells, in turn, activate B-cells to produce antibody that may lead tothe destruction of the cell. Thus, recognition of a newly administeredcell or tissue as non-self is one aspect of the cascade of events givingrise to transplant rejection.

Autologous cell therapies are attractive, and commonly used, as the“self” immune profile of administered cells/tissues will rarely elicitan immune response upon re-administration to the donor/recipient.Autologous transplant with embryonic tissue is not feasible in the vastmajority of cases, as harvesting embryonic tissue for later use in thesame individual is technologically and temporally challenging. Thus,embryonic cells are typically allogeneic with respect to the recipient.As a result, rejection of transplanted embryonic cells may be asignificant concern. Additionally, the pluripotency of embryonic stemcells does not guarantee differentiation of implanted/administered cellsinto cells related to the target tissue. In other words, an embryonicstem cell implanted into the heart may not necessarily yield hearttissue, but rather may yield other, unwanted cell types or result interatoma formation.

According to several embodiments of the present invention, adult stemcells, whether allogeneic, autologous, xenogeneic, or syngeneic developinto cell types closely related to the originating tissue type. In otherwords, adult cardiac regenerative cells will differentiate into cardiacrelated cell types, such as cardiomyocytes or cardiac endothelial cellsand vasculature, among other cardiac cell types. Several embodiments ofthe invention are especially advantageous because the risk that adultstem cells will develop into undesired cell types is less than whenembryonic stem cells are used and can be further reduced by isolatingadult stem cells from the tissue that is to be treated or repaired.

The determination of whether to use autologous or allogeneicregenerative cells may not be driven primarily by temporal difficultieswith respect to cell isolation, but by other clinical implications.Tissue collection for isolation of adult regenerative cells is commonlyaccomplished by simple biopsy procedures. In many cases, the currentstate of a donor's tissue is a determining factor on whether to useautologous or allogeneic regenerative cells. For example, a donor whosuffered from extensive tissue damage may have insufficient or non-idealtissue from which to isolate regenerative cells. In such cases,allogeneic regenerative cells may present a preferred alternative.

Allogeneic regenerative cells do present the possibility of immunerejection by the recipient, thereby potentially limiting the long termsurvival of the administered regenerative cells. However, allogeneicregenerative cells also present numerous benefits. They can be harvestedfrom healthy donors, expanded in culture, and stored for future use,meaning there is a ready availability of regenerative cells for use intherapies. In the cardiac context in particular, this ready supply ofstored regenerative cells would enable administration of cells in thecritical post-injury period, where beneficial therapeutic outcomes maybe maximal.

Additionally, because the donor and recipient are distinct from oneanother, allogeneic regenerative cells can be obtained from healthytissue of a healthy donor. This may improve the survival of theregenerative cells in long term storage, as well as during thepost-administration period. In addition, regenerative cells from ahealthy donor may simply induce a more robust and positive therapeuticeffect than regenerative cells taken from a recipient in a state ofcompromised health. Additionally, even though adult regenerative cellscan be isolated via a simple biopsy procedure, allogeneic transplantsdecrease risk to the recipient, such as infection risk, as the recipientneed not undergo the tissue isolation procedure. Moreover, theseadvantages have the potential to increase the number of recipients thatcan receive allogeneic transplants and simultaneously reduce the cost ofproviding such therapies.

Several embodiments described herein provide for methods of isolating,methods of culturing, methods of preparing, and methods of introducingregenerative cells into a recipient in need of amelioration of thesymptoms associated with and/or treatment of a cardiac condition. Inseveral embodiments, the regenerative cells are cardiac tissue-derived.In certain such embodiments, the regenerative cells are adultregenerative cells. As used herein, the term adult shall be given itsordinary meaning and shall also refer to all stages of life extendingfrom birth to death, (e.g., adult cells are non-embryonic/non-fetalcells). Additionally, in several embodiments, adult cells or adulttissues also refer to cells or tissues collected after death of an adultindividual.

In several embodiments the regenerative cells are allogeneic. In someembodiments, allogeneic regenerative cells are harvested from healthydonors, expanded in culture, and stored for future use. In certain suchembodiments, a pool of allogeneic regenerative cells for acute therapyis available. Thus, in some embodiments, an allogeneic source ofregenerative cells and allogeneic transplant reduces overall risk to therecipient. In some embodiments, the allogeneic regenerative cells arestored in a manner that allows for rapid preparation and administrationto a recipient in need of cell therapy. In certain embodiments, suchregenerative cells are administered to a recipient as soon as possibleafter the recipient has suffered an adverse cardiac event. However, inother embodiments, the regenerative cells are administered over a periodof time, multiple times, or after a certain period of time, depending onthe severity and nature of an adverse cardiac event.

In several allogeneic embodiments, the donor regenerative cells have notbeen immunologically matched with respect to the subject. In otherembodiments, donor regenerative cells are known to be immunologicallymismatched with respect to the recipient. In such embodiments, the donorregenerative cells are mismatched (with respect to the recipient) at oneor more HLA antigens. However, in certain embodiments, the degree ofmismatch does not necessarily predict a severity of immune response. Inother words, in some embodiments involving a donor and recipient havinga larger degree of immunological mismatch there is a less severe immuneresponse as compared to a donor and recipient who are moreimmunologically similar.

In several embodiments the regenerative cells are autologous. In someembodiments, autologous regenerative cells are harvested from a donor ata point when the donor is healthy, expanded in culture, and stored forfuture use. In certain such embodiments, a pool of autologousregenerative cells for personally tailored cell therapy is available. Incertain embodiments, the autologous regenerative cells are stored in amanner that allows for rapid preparation and re-administration to thedonor/recipient when the donor/recipient is in need of cell therapy. Incertain embodiments, such regenerative cells are administered to thedonor/recipient as soon as possible after the recipient has suffered anadverse cardiac event. However, in other embodiments, the regenerativecells are administered over a period of time, multiple times, or after acertain period of time, depending on the severity and nature of anadverse cardiac event.

In several embodiments the stem cells are xenogeneic. In certain suchembodiments, a large pool of xenogeneic regenerative cells isolated froma donor organism is available. In certain embodiments, the xenogeneicregenerative cells are stored in a manner that allows for rapidpreparation and administration to the recipient when the recipient is inneed of cell therapy. In certain embodiments, such regenerative cellsare available and ready for administration to the recipient as soon aspossible after the recipient has suffered an adverse cardiac event.However, in other embodiments, the regenerative cells are administeredover a period of time, multiple times, or after a certain period oftime, depending on the severity and nature of an adverse cardiac event.

In several embodiments of the invention, the regenerative cells aresyngeneic. In some embodiments, syngeneic regenerative cells areharvested from a healthy donor, expanded in culture, and stored forfuture use in either the donor (in which case the transplant would beautologous) or a genetically related recipient. In certain suchembodiments, a pool of syngeneic regenerative cells for cell therapytailored to a specific genetic and/or immunological background isavailable. In certain embodiments, the syngeneic regenerative cells arestored in a manner that allows for rapid preparation andre-administration to the donor/recipient when the donor/recipient is inneed of cell therapy. In certain embodiments, such regenerative cellsare administered to the donor/recipient as soon as possible after therecipient has suffered an adverse cardiac event. However, in otherembodiments, the regenerative cells are administered over a period oftime, multiple times, or after a certain period of time, depending onthe severity and nature of an adverse cardiac event.

As used herein, the term “adverse cardiac event” shall be given itsordinary meaning and shall also be read to include, but not be limitedto myocardial infarction, ischemic cardiac tissue damage, congestiveheart failure, aneurysm, atherosclerosis-induced events, cerebrovascularaccident (stroke), and coronary artery disease.

In several embodiments, the regenerative cells, whether allogeneic,autologous, xenogeneic, or syngeneic, are multipotent. In certainembodiments, the regenerative cells advantageously present a decreasedrisk of abnormal tissue or teratoma formation, and depending on thetransplant type, a reduced risk of immunological rejection. Despitethese advantages, in certain other embodiments, pluripotent regenerativecells are used.

The Role Model Effect—Signals and Cellular Recruitment Induced byRegenerative Cells

Depending on the cell types involved, and the type of signal to beconveyed, multiple varieties of cell signaling are used. Autocrinesignaling involves the generation of a signal that acts back on the samecell (or same type of cell). In contrast paracrine signaling involves atarget cell which is near, but distinct from the signal-releasing cell.Among other events, paracrine signaling is involved in allergic andimmune responses, tissue growth and repair, and blood clotting.

The beneficial effects which are observed upon administration of thecells to a mammal may be due to the cells per se in some embodiments.For example, in some embodiments the engraftment of cells produces afavorable outcome. However, in some embodiments, the beneficial effectsare due to products which are expressed by the cells (e.g., a surfacemarker that interacts with host tissue) or secreted, released orotherwise delivered to the recipient tissue by the cells. In severalembodiments cytokines or chemokines or other diffusible factors,including but not limited to paracrine factors (e.g., growth factors)stimulate resident cells to grow, reproduce, or perform better. In someembodiments, the combination of the cells and diffusible factors providethe beneficial effects.

In several embodiments, the regenerative cells administered to arecipient produce paracrine signals that affect the surrounding targettissue during or after administration. However, in several embodiments,the direct administration of regenerative cells is not necessary, inthat the culturing of isolated regenerative cells results in release ofthe paracrine signaling molecules into the culture media, which can thenbe harvested and administered in place of the regenerative cells. Insome embodiments, regenerative cells and their paracrine signalingmolecule-enriched media are co-administered. In other embodiments, stemcells and their paracrine signaling molecule-enriched media aresequentially administered. In still other embodiments, regenerativecells (in a pharmaceutically acceptable carrier) are administered alone.

In several embodiments, paracrine signals from the administeredregenerative cells have multiple positive effects on the surroundingtarget tissue. In certain embodiments, the effects of the paracrinesignals persist, even after the administered regenerative cells are nolonger viable. In other words, the regenerative cells create a “RoleModel” effect or a “butterfly effect”, in that they set in motion acascade of events that carries on, even when the regenerative cells areno longer present. In some embodiments, the paracrine signals generatedimprove the viability of the surrounding target tissue (e.g. signals arepro-survival). In certain such embodiments, paracrine signals act onboth damaged and healthy target tissue. In some embodiments, paracrinesignals enhance the recovery of damaged cells in the target tissue. Insome embodiments, paracrine signals enhance the function of damagedand/or healthy cells in the target tissue. In some embodiments,paracrine signals induce the regeneration of new target tissue. In someembodiments, paracrine signals enhance the recovery of damaged cells inthe target tissue.

In several embodiments, the paracrine signals reduce the amount ofinduced programmed cell death (apoptosis). In some embodiments,reduction in apoptosis is manifest by a reduction in the expression ofcertain apoptotic markers in the target tissue. In other embodiments,anti-apoptotic markers are increased. In some embodiments, apoptoticmarkers are simultaneously reduced while anti-apoptotic markers areincreased. In some embodiments, reduction in apoptosis is manifest by areduction in the number of cells in target tissue that are permeable tocertain molecules (i.e. fewer cells have the characteristic plasmamembrane damage associated with apoptosis). In some embodiments,reduction in apoptosis in the target tissue occurs rapidly afteradministration of regenerative cells. In other embodiments, reductionapoptosis in the target tissue occurs after several hours or dayspost-administration of regenerative cells. In some such embodiment,reductions in apoptosis are detected between about 24 and about 72 hourspost-administration. In some embodiments, apoptosis is reduced after 1week. In other embodiments, apoptosis remains low for several weeks. Insome embodiments, apoptosis is reduced by up to 20%. In someembodiments, apoptosis is reduced by about 20%. In other embodiments,apoptosis is reduced by about 30%. In some embodiments, apoptosis isreduced by about 35%. In some embodiments apoptosis is reduced by about40%. In certain embodiments, apoptosis is reduced by about 20-30%,including 21, 22, 23, 24, 25, 26, 27, 28, and 29%.

In several embodiments, paracrine signals induce formation of new bloodvessels, which thereby improves function and/or survival of the targettissue (e.g., signals are pro-angiogenic). In some embodiments, newblood vessel formation is manifest by an increase in the length ofexisting vessels (e.g., into the infarcted area). In some embodiments,new blood vessel formation is manifest by an increase in the density ofvessels or in an area of tissue. In some embodiments, vessel densityincreases by up to about 4-fold (as compared to damaged tissue notreceiving regenerative cells). In other embodiments, vessel densityincreases by about 2-fold. In some embodiments, vessel density increasesby about 3-fold. In some embodiments, vessel density increases by about1.5-fold. In certain embodiments, vessel density increases by about anamount ranging from about 1.1 to 2.5-fold, including 1.2, 1.4, 1.6, 1.8,2.2, and 2.4-fold.

In several embodiments, the paracrine signals generating the new bloodvessels are carried to more remote locations in the target tissue, andinduce positive effects in the remote tissue. In some embodiments,paracrine signals recruit endogenous stem cells from the surroundingtissue. In certain other embodiments, paracrine signals from theadministered stem cells initiate a signaling cascade, causing otherlocal cells to generate additional paracrine signals. In still otherembodiments, paracrine signals from the administered regenerative cellsact both on endogenous cells in a paracrine manner as well in anautocrine manner on the stem cells themselves. In several embodimentswherein two or more paracrine signals are generated, the signalsfunction in a synergistic manner to generate one or more of the positiveeffects described herein. Thus, it shall be appreciated that theadministration of regenerative cells, in several embodiments asdescribed above, yield positive effects in the target tissue througheither a direct effect (e.g., tissue regeneration), indirect effect(e.g., increase blood supply to new and endogenous tissue), or acombination thereof.

In several embodiments, the type of regenerative cells administeredplays a role in determining one or more of the type, duration, orintensity of paracrine signals generated. For example, in certainembodiments, a first type of regenerative cells may release a particularamount of a first paracrine signal upon administration, while in otherembodiments, a different type of regenerative cells may release less ofthe first paracrine signal, and more of a second (or additional)paracrine signal. It shall be appreciated that while the types ofregenerative cells are genetically related, their differing structures,compositions, and/or state of differentiation make it possible that theparacrine signals released by one type of regenerative cells are quitedifferent than those released by its derivatives. For example, themicroenvironment surrounding certain stem cells is known to play a rolein the regulation of stem cells. Thus, differentiation of embryonic stemcells is both spatially and temporally regulated by the distinctenvironments, or “cellular niches” created during development. Asdiscussed above, removal of an embryonic stem cell and placement of thecell in a distinct niche disrupts the signals of the nativemicroenvironment, often resulting in teratoma formation.

Certain regenerative cells have a multicellular, 3-dimenensionalstructure, thus creating the possibility of numerous microenvironmentsor niches within the regenerative cells. For example, based on the3-dimensional structure of some regenerative cells, a gradient ofcellular oxygen exists that decreases from the outer to the inner celllayers. Thus in some embodiments, certain cells in the inner layers arestimulated to release paracrine signals by the level of hypoxia thecells experience in their immediate environment. In some embodiments,the close physical proximity of certain cell types within theregenerative cells create a contact-based microenvironment, whichsubsequently directs the function and fate of the regenerative cells. Inseveral embodiments, the paracrine signals produced by the cellscomprising the regenerative cells create a distinct microenvironmentwhich subsequently directs the function and fate of the regenerativecells. Thus, in certain embodiments, regenerative cells having a 3-Dstructure are preferred. However, non-3-dimensional regenerative cellsare used in other embodiments.

In several embodiments, certain regenerative cells release more or lessof the paracrine signals released by other regenerative cells, and mayrelease one or more additional paracrine signals not released by otherregenerative cells. In some such embodiments, each type of regenerativecells has the capacity to generate any or all of the paracrine signalsgenerated by the other types (and vice versa) but is not stimulated todo so.

In several embodiments, the generation of paracrine signals fromregenerative cells varies over time (either during in vitro culture orin vivo, post-administration). In some embodiments, generation ofparacrine signals continues for at least a week after administration orculturing begins. In other embodiments, generation of paracrine signalscontinues for two, three or four weeks, several months, or for severalyears. In several embodiments, the paracrine signals, either alone or incombination with the signals generated by endogenous tissue, promote theengraftment and/or long term survival of the administered regenerativecells. In other embodiments, the engraftment and survival are relativelyshort-lived, but the resultant effects are long-term. In someembodiments, the survival of administered regenerative cells is due totheir ability to differentiate into multiple types of cardiac tissue,thus efficiently adapting to the local environment. In some embodiments,the adaptability of regenerative cells functions in combination withparacrine signals to effectuate the survival of the administered cells.

In several embodiments, the administration of regenerative cells resultsin engraftment of the regenerative cells into host tissue. In someembodiments, as discussed herein, the amount of detectable engraftmentof the regenerative cells changes over time. In some embodiments, ahigher degree of regenerative cells have engrafted a short period oftime after administration as compared to later times afteradministration (i.e. engrafted numbers decrease). In some embodiments,engraftment peaks at an intermediate time point. In some embodiments,the type of regenerative cells used (i.e., allogeneic v. autologous,etc.) is a factor in engraftment. In some embodiments, allogeneicregenerative cells engraft as well as other types, while in otherembodiments, allogeneic regenerative cells show lesser engraftment. Insome embodiments, engraftment of allogeneic regenerative cells isequivalent to that of other regenerative cells types at a first timepoint, but decreases more rapidly post-administration. However, inseveral embodiments, the functional effect of allogeneic regenerativecells is equivalent, or greater than, that of other cell types, despitethe lesser degree of engraftment. In certain embodiments, engraftment iscorrelated with survival of regenerative cells. In some embodiments, theadministered regenerative cells survive for several dayspost-administration. In some embodiments, administered regenerativecells survive for about a week to about two weeks. In some embodiments,administered regenerative cells survive for several weeks, or one, two,three or more months. As discussed above, in certain embodiments, theeffects of the paracrine signals, and in some embodiments the signalsthemselves, persist after administered regenerative cells are no longerviable. In several embodiments, this “butterfly effect”, the persistenceof the signals that result from the engrafted cells, despite the limitedterm of the engraftment, is responsible for the long-term beneficialanatomical and functional recovery of the cardiac tissue.

In several embodiments, the paracrine signals comprise one or moregrowth factors, hormones, cytokines, or other signaling molecule thatare released from the administered regenerative cells. In certainembodiments the paracrine signals comprise one or more of the followingsignaling molecules: ENA-78, G-CSF, GM-CSF, GRO, GRO-alpha, I-309, IL-1alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-13, IL-15, interferon gamma, MCP-1, MCP-2, MCP-3, M-CSF, MDC,MIG, MIP-1 beta, MIP-1 delta, RANTES, SCF, SDF-1, TGF-beta 1, TNF-beta,EGF, IGF-1, angiogenin, oncostatin M, thrombopoeitin, VEGF, PDGF-BB,leptin, BDNF, BLC, Ck beta 8-1, eotaxin, eotaxin-2, eotaxin-3, FGF-4,FGF-6, FGF-7, Flt-3 ligand, fractalkine, GCP-2, GDNF, HGF, IGFBP-1,IGFBP-2, IGFBP-3, IGFBP-4, IL-16, IP-10, LIF, LIGHT, MCP-4, MIP-3 alpha,NAP-2, NT-3, NT-4, osteopontin, osteoprogenerin, PARC, PIGF, TGF beta 2,TGF beta 3, TIMP-1 and TIMP-2. In several preferred embodiments, theparacrine signals comprise one or more of VEGF, HGF, and IGFI. In someembodiments, a single paracrine signal is responsible for the beneficialtherapeutic effects, while in other embodiments, one or more paracrinesignals work synergistically to produce the effects. As discussed above,several embodiments described herein reduce the risk of teratomaformation. In certain embodiments, the paracrine signals from theregenerative cells (or those induced in the target tissue) reduce,minimize, and/or eliminate the risk of teratoma formation.

In several embodiments, various types of regenerative cells havedifferent paracrine potencies. In other embodiments, the paracrinepotency of a single type of regenerative cells varies over time. In someembodiments, regenerative cells express one or more receptors for theparacrine signals generated by the regenerative cells. As such, in someembodiments the regenerative cells act in a paracrine manner on otherco-administered regenerative cells. In some embodiments, theregenerative cells act in a paracrine manner on endogenous tissue. Instill other embodiments, the regenerative cells act in an autocrinemanner. In some embodiments, the regenerative cells express one or moreof the KDR, Met, and IGFI receptors (the receptors for receptors forVEGF, HGF and IGFI, respectively).

In several embodiments, endogenous cells are recruited by theadministration of the regenerative cells. In some embodiments, thepresence of the administered regenerative cells induces the recruitmentof endogenous cells. In other embodiments, paracrine signals releasedfrom the regenerative cells induce the recruitment of endogenous cells.In still other embodiments, the presence of the regenerative cells andthe paracrine signals produced thereby work in combination to induce therecruitment of endogenous cells.

In several embodiments, the recruited endogenous cells improve theviability of the surrounding target tissue. In certain embodiments, therecruited cells engraft into the damaged tissue and generate new,healthy tissue. In certain embodiments, the recruited endogenous cellsgenerate paracrine signals that act on both damaged and healthy targettissue. In some embodiments, these paracrine signals enhance therecovery of damaged cells in the target tissue. In certain embodiments,these paracrine signals reduce the amount of programmed cell death. Insome embodiments, paracrine signals from the recruited endogenous cellsenhance the function of damaged and/or healthy cells in the targettissue. In some embodiments, these paracrine signals induce theregeneration of new target tissue. In some embodiments, these paracrinesignals enhance the recovery of damaged cells in the target tissue. Insome embodiments, paracrine signals from the recruited endogenous cellsinduce formation of new blood vessels, which thereby improves functionand/or survival of the target tissue. In certain such embodiments, theseparacrine signals generating the new blood vessels are carried to moreremote locations in the target tissue, and induce positive effects inthe remote tissue.

In certain other embodiments, paracrine signals from the recruitedendogenous cells initiate a signaling cascade, causing other local cellsto generate additional paracrine signals. In still other embodiments,paracrine signals from the recruited endogenous cells act both on otherendogenous cells in a paracrine manner as well in an autocrine manner onthe recruited endogenous cells themselves. In several embodimentswherein two or more paracrine signals are generated, the signalsfunction in a synergistic manner to generate one or more of the positiveeffects described herein. Thus, it shall be appreciated that therecruitment of endogenous cells, in several embodiments as describedabove, yield positive effects in the target tissue through either adirect (e.g., tissue regeneration of increased function traceable to theendogenous cells), indirect effect (e.g., paracrine signals induceincreased blood supply to new and endogenous tissue), or a combinationthereof.

In several embodiments, the regenerative cells are used in thepreparation of a medicament. In some embodiments, the medicament issuitable for administration to an individual having damaged or diseasedtissue, in particular damaged or diseased cardiac tissue. In someembodiments, administration of the medicament results in one or more ofalleviation of symptoms of a cardiac disease, improvement in cardiacfunction, and/or regeneration of cardiac tissue in the recipientindividual.

Methods of Harvesting Cardiac Tissue and Producing Regenerative Cells

It shall be appreciated that the term “regenerative cells” as usedherein refers to each of the mixed cell populations described herein(cardiospheres) and derivatives thereof (CDCs and IICSps). In describingthe isolation methods, and the methods to produce derivatives of certainregenerative cells, the individual type of regenerative cells is usedfor clarity.

Donor tissue may be obtained from embryonic or adult sources. Adultsources are preferred for several embodiments. In some embodiments,donor cardiac tissue is obtained during a surgical procedure, such asbypass surgery. In other embodiments, donor tissue is obtained during apercutaneous endomyocardial biopsy procedure. In still otherembodiments, large quantities of tissue are obtained from recentlydeceased organ donors, processed, and stored for use in allogeneictransplants. While a typical human adult heart weighs about 200 to 300g, sufficient amounts of regenerative cells can be obtained from cardiactissue samples of about 1 mg to about 50 mg. In some embodiments, themass of the cardiac tissue sample is about 25 mg or less. The tissuesample may be obtained from a variety of locations in the heart,including but not limited to, the crista terminalis, the rightventricular endocardium, the right ventricular septum, the septal orventricle wall, the atrioventricular groove and the right and leftatrial appendages.

In one embodiment, the tissue sample is obtained from donor hearts thatare transplant quality, but are unable to be transplanted into a humanpatient (e.g., because of unavailability of a matched recipient, etc.).In one embodiment, the tissue sample is obtained from hearts that arenot transplant quality because (e.g., of tissue damage in the donorheart, the age of the heart, suspected cardiovascular disease in thedonor, etc.). In several embodiments, the entire heart is processed toobtain a tissue sample for culture. In other embodiments, tissue isextracted from one or more of the following: atria, atrial appendagesand apex.

According to several embodiments, percutaneous endomyocardial biopsyspecimens are harvested using the following procedure. Under localanesthesia, a guide catheter is introduced into a vein, such as thejugular vein, in the patient's neck if tissue samples are to be takenfrom the right ventricle. Alternatively, the guide catheter can beintroduced into an artery if tissue samples are to be taken from theleft ventricle. The guide catheter is guided to the heart with the aidof visualization provided by a standard imaging technique, such asfluoroscopy. Once the guide catheter is in place, a bioptome can beintroduced into the guide catheter and threaded to the heart. Once thebioptome is within the heart, the flexible distal end of the bioptomecan be manipulated by the surgeon to extract a tissue sample from thedesired location. The bioptome can be removed from the patient so thatthe tissue sample can be retrieved and then the bioptome can bereintroduced so that another sample can be taken from the same ordifferent location. In another embodiment, the bioptome can extractmultiple samples before being withdrawn, thereby reducing the timeneeded to collect the tissue samples.

In one embodiment, the invention comprises harvesting a piece ofmyocardial tissue about 0.25-about 1 cm in length and width through acatheter placed in the jugular vein of a subject under local anesthesia.In one embodiment, the weight of the sample is about 0.25-about 1 gram.The heart biopsy sample is then cultured over a period of about 3 toabout 6 weeks (e.g., about 4 weeks) until approximately 10 to 25 millioncells are available for implantation into the coronary arteries. Asdisclosed herein, the biopsy sample may be obtained from a first subjectand then implanted into the same subject. Alternatively, the biopsysample may be obtained from a first subject and then implanted into adifferent subject.

In several embodiments, the processing of a biopsy sample yieldsregenerative cells that comprise a mixed population of cells thatcomprises, for example, stem cells, cardiac cells, and/or vascularcells, among other cell types. In some embodiments, the mixed populationof cells expresses various stem cell markers. In some embodiments, stemcells of the mixed population may be identified by expression of stemcell-related markers including one or more of CD-105, CD90, CD34, Sca-1,and c-kit, among others. In certain embodiments, the stem cells do notexpress one or more of the stem cell markers identified above. Incertain embodiments, the stem cells are CD45 negative. In someembodiments, the vascular cells of the mixed cell population express atleast one of KDR, flk-1, CD31, von Willebrand factor, Ve-cadherin, andsmooth muscle alpha actin, among others.

In vitro, the mixed cell populations are clonogenic and can give rise toimmature cardiomyocytes (heart muscle cells) and endothelial and smoothmuscle cells (blood vessel components). In addition, in someembodiments, CDCs may be grown on a solid surface to produce a second(or greater) generation of cardiospheres (IICSps).

In one embodiment of the invention, regenerative cells are isolated andcultured as according to the schematic in FIG. 1. Briefly, cardiactissue samples are weighed, cut into small fragments and cleaned ofgross connective tissue, and washed in a sterile solution, such asphosphate-buffered saline. In some embodiments, the tissue fragments areat least partially digested with protease enzymes such as collagenase,trypsin, and the like. In certain embodiments, the digested pieces areplaced in primary culture as explants on sterile tissue culture disheswith a suitable culture media. The digested pieces of tissue range insize from about 0.1 mm to about 2.5 mm. In several embodiments, thedigested pieces of tissue range 0.25 mm to about 1.5 mm. Smaller orlarger pieces of tissue can be used in other embodiments. The tissueculture dish and culture media are selected so that the tissue fragmentsadhere to the tissue culture plates. In some embodiments, the tissueculture plates are coated with fibronectin or other extracellular matrix(ECM) proteins, such as collagen, elastin, gelatin and laminin, forexample. In other embodiments, the tissue culture plates are treatedwith plasma. In certain embodiments, the dishes are coated withfibronectin at a final concentration of from about 10 to about 50 μg/mL.In still other embodiments, the fibronectin dishes are coated withfibronectin at a final concentration of from about 20 to 40 μg/mL, withstill other embodiments employing a final fibronectin concentration ofabout 25 μg/mL.

In certain embodiments, the base component of the complete explantmedium comprises Iscove's Modified Dulbecco's Medium (IMDM). In someembodiments, the culture media is supplemented with fetal calf serum(FCS) or fetal bovine serum (FBS). In certain embodiments, the media issupplemented with serum ranging from 5 to 30% v/v. In other embodiments,the culture media is serum-free and is instead supplemented withspecific growth factors or hydrolyzed plant extracts. In otherembodiments, the media us supplemented with serum, but no additionalexogenous growth factors. In yet other embodiments, the media is furthersupplemented with antibiotics, essential amino acids, reducing agents,or combinations thereof. In one embodiment, the complete explant mediumcomprises IMDM supplemented with about 20% fetal bovine serum, about 50μg/mL gentamicin, about 2 mM L-glutamine, and about 0.1 mM2-mercaptoethanol. In some embodiments, the explant media is changedevery 2-4 days while the explants culture.

The tissue explants are cultured until a layer of stromal-like cellsarise from adherent explants. This phase of culturing is furtheridentifiable by small, round, phase-bright cells that migrate over thestromal-cells. In certain embodiments, the explants are cultured untilthe stromal-like cells grow to confluence. At or before that stage, thephase-bright cells are harvested. In certain embodiments, phase-brightcells are harvested by manual methods, while in others, enzymaticdigestion, for example trypsin, is used. The phase-bright cells may betermed cardiosphere-forming cells, and the two phrases are usedinterchangeably herein.

Cardiosphere-forming cells may then be seeded on sterile dishes andcultured in cardiosphere media. In certain embodiments, the dishes arecoated with poly-D-lysine, or another suitable natural or syntheticmolecule to deter cell attachment to the dish surface. In otherembodiments, for example, laminin, fibronectin, poly-L-orinthine, orcombinations thereof may be used.

In certain embodiments, the base component of the cardiosphere mediumcomprises Iscove's Modified Dulbecco's Medium (IMDM). In someembodiments, the culture media is supplemented with fetal calf serum(FCS) or fetal bovine serum (FBS). In certain embodiments, the media issupplemented with serum ranging from 5 to 30% v/v. In other embodiments,the culture media is serum-free and is instead supplemented withspecific growth factors or hydrolyzed plant extracts. In certain otherembodiments, the media is further supplemented with antibiotics,essential amino acids, reducing agents, or combinations thereof. In oneembodiment the cardiosphere medium comprises IMDM supplemented withabout 10% fetal bovine serum, about 50 μg/mL gentamicin, about 2 mML-glutamine, and about 0.1 mM 2-mercaptoethanol.

According to one embodiment, cardiospheres will form spontaneouslyduring the culturing of the cardiosphere forming cells. Cardiospheresare recognizable as spherical multicellular clusters in the culturemedium. Cells that remain adherent to the poly-D-lysine-coated dishesare discarded. In certain embodiments, the cardiospheres are collectedand used to seed a biomaterial or synthetic graft. In other embodiments,the cardiospheres are further cultured on coated cell culture flasks incardiosphere-derived stem cell (CDC) medium.

In some embodiments used to culture cardiospheres into CDCs, theculturing flasks are fibronectin coated, though in other embodimentsother cellular attachment promoting coatings are employed. The culturedcardiospheres attach to the surface of the flask and are expanded as amonolayer of CDCs. CDC medium comprises IMDM, and in certain embodimentsis supplemented with fetal calf serum (FCS) or fetal bovine serum (FBS).In some embodiments, the media is supplemented with serum ranging from 5to 30% v/v. In other embodiments, the culture media is serum-free and isoptionally supplemented with specific growth factors or hydrolyzed plantextracts. In certain other embodiments, the media is furthersupplemented with antibiotics, essential amino acids, reducing agents,or combinations thereof. In one embodiment, the CDC medium comprisesIMDM supplemented with about 10% fetal bovine serum, about 2 mML-glutamine, and about 0.1 mM 2-mercaptoethanol. CDCs may be repeatedlypassaged by standard cell culture techniques. In some embodiments, CDCsare detached from the culturing surface and plated onpoly-D-lysine-coated dishes to form a second generation of cardiospheres(IICSps). This process of generating cardiospheres followed by CDCsfollowed by a subsequent generation of cardiospheres may be repeated asneeded to expand the population of any of the regenerative cell types.

Administration of Regenerative Cells

In several embodiments, regenerative cells are administered torecipients systemically. In some such embodiments, the systemicallyadministered cells migrate to the recipient's heart, particularly to thearea of damaged tissue. In several embodiments, regenerative cells aredelivered systemically via an intravenous route. In several embodiments,the regenerative cells are delivered locally. In certain embodiments,local delivery is achieved by direct myocardial injection. In certainembodiments, local delivery is achieved via a biopsy procedure. In someembodiment, delivery is accomplished during a surgical procedure.Delivery may be accomplished with specific injection site guidance incertain embodiments. In one embodiment, NOGA is employed. In certainembodiments, regenerative cells are delivered alone, while in otherembodiments regenerative cells are delivered in associated with anadditional therapeutic agent. In still other embodiments, the paracrineagents produced by regenerative cells are administered, either alone orin conjunction with the regenerative cells.

In certain embodiments, the regenerative cells are used to seed abiomaterial or synthetic graft. In certain embodiments, the graftcomprises a biocompatible biomaterial such as hyaluronan, alginate, orfibrin. In some embodiments, the biomaterial or synthetic graft isinjectable. In other embodiments, the biomaterial or synthetic graft ispainted or directly placed onto the target tissue.

In several embodiments, the regenerative cells are delivered at a doseof about 1×10⁵ to about 1×10⁷ regenerative cells per kilogram of bodyweight of the recipient. However, in some embodiments, lower numbers ofcells may be used, due to the butterfly effect described herein (e.g.,the persistence of positive effects on the target tissue even after someor all of the delivered cells are removed by host mechanisms).

Immune Responses and Functional Effects of Regenerative Cells

As discussed above, the various transplant types, and cells used in eachhave advantages and disadvantages. Autologous transplants areadvantageous due to limited risk for immune rejection of transplantedcells. Thus in some embodiments, autologous regenerative cells are used.However, in certain instances, autologous transplants are expensive, andsomewhat time consuming (since tissue must be harvested, processed intoregenerative cells, and re-administered), especially in circumstanceswhere the donor/recipient requires immediate administration of therapy(e.g., they are within a critical post-injury period, typically within afew hours of an adverse event). Therefore, an ideal cellular therapywould be available “off the shelf”, and preferably would not inducesevere immune responses in the recipient, or at least be able toinitiate or yield a therapeutic response despite an immune response.Thus, in some embodiments, allogeneic regenerative cells are used.

Immune responses mounted by a recipient may lead to rejection oftransplanted cells that are immunologically distinct. Such cells, forexample, allogeneic regenerative cells, may be rejected through a director indirect pathway. Direct rejection by the recipient involves eitherantigen presenting cells (APCs) that were transplanted from the donor ortheir donor APC precursor cells that have differentiated into APCs aftertransplantation. When a recipient T-cell (also known as T-lymphocytes,immune cells that play a central role in cell-mediated immunity)recognizes a donor APC as “non-self” (via expression of donor HLAmolecules or other donor-derived antigens), the recipient T-cell becomesactivated, recruits other recipient immune co-stimulatory moleculesbecome involved (such as CD80 or CD86 with CD28, and CD40 with CD40ligand), and an immune response is initiated.

Indirect rejection may occur due to the “shedding” various antigens fromtransplanted donor cells or tissues. These donor antigens are taken upby recipient APCs, and subsequently presented to recipient T cells. Thiscan result in the activation of donor-reactive recipient T cells, whichthen initiate an immune response.

In several embodiments, an immune response is initiated againsttransplanted allogeneic regenerative cells. In several embodiments, thegrade of any immune response is higher than the grade of a correspondingautologous or syngeneic transplant, however, in such embodiments, theseverity of the immune response does not eliminate all of thetransplanted regenerative cells, or their effect on the target tissue.In some embodiments, the grade of immune response in an allogeneictransplant scenario is equivalent to or less than that of an autologousor syngeneic transplant. In certain embodiments, as discussed herein,some of the administered regenerative cells survive for several dayspost-administration. In some embodiments, some of the administeredregenerative cells survive for about a week to about three weeks. Insome embodiments, a local immune effect is initiated againsttransplanted regenerative cells. In certain such embodiments, the localimmune response does not destroy or render non-functional thetransplanted regenerative cells until after a series of signal or eventshave been initiated that lead to a beneficial therapeutic effect. Insome embodiments, a systemic immune response is initiated againsttransplanted regenerative cells. In certain such embodiments, thesystemic immune response does not destroy or render non-functional thetransplanted regenerative cells until after a series of signal or eventshave been initiated that lead to a beneficial therapeutic effect. Instill other embodiments, there is little or no systemic immune response.Thus, in certain embodiments, the effects initiated by theadministration of the regenerative cells persist after some, or all, ofthe administered regenerative cells are no longer viable.

In several embodiments, the transplant of regenerative cells (regardlessof transplant type) results in a beneficial therapeutic effect in therecipient. As discussed above, the beneficial therapeutic effect maycomprise one or more of treatment of symptoms of a cardiac disease,improvement in cardiac function, and/or regeneration of cardiac tissuein the recipient.

In several embodiments, transplanted regenerative cells result insignificantly increased fractional area. In some embodiments, fractionalarea is increased by about 5%. In some embodiments, fractional area isincreased by about 10%. In some embodiments, fractional area isincreased by about 15%. In some embodiments, fractional area isincreased by about 20%. In some embodiments, fractional area isincreased by about 5-10%, including 6, 7, 8, and 9%. In someembodiments, fractional area is increased by about 10-20%, including 11,12, 13, 14, 15, 16, 17, 18, and 19%. In still additional embodiments,greater increases are realized.

In several embodiments, transplanted regenerative cells result insignificantly increased ejection fraction. In some embodiments, ejectionfraction is increased by about 5%. In some embodiments, ejectionfraction is increased by about 10%. In some embodiments, ejectionfraction is increased by about 15%. In some embodiments, ejectionfraction is increased by about 20%. In some embodiments, ejectionfraction is increased by up to about 25%. In some embodiments, ejectionfraction is increased by about 6-12, 12-18, or 19-25% In someembodiments, ejection fraction is increased by about 5-20%, including 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19%.

In several embodiments, transplanted regenerative cells result in adecrease in infarct size (i.e. an increase in viable tissue in andaround the infarct). In some embodiments, infarct size is reduced byabout 5%. In some embodiments, infarct size is reduced by about 10%. Insome embodiments, infarct size is reduced by about 15%. In someembodiments, infarct size is reduced by about 1-3, 4-7, or 7-11%. Insome embodiments, infarct size is reduced by 5-15%, including 6, 7, 8,9, 10, 11, 12, 13, and 14%.

In several embodiments, the beneficial therapeutic effect oftransplanting regenerative cells (regardless of the transplant type)lasts for several weeks. In some embodiments, the beneficial therapeuticeffect last for up to three weeks. In some embodiments, the beneficialtherapeutic effect lasts for a period of time from about 3 weeks toabout 12 weeks. In some embodiments, the beneficial therapeutic effectlasts for about 3 months to about 1 year. In still other embodiments,the beneficial therapeutic effect lasts for several years.

In several embodiments of the invention, a method of treating an adversecardiac event is provided, wherein the method comprises implantingregenerative cells into a recipient patient. The regenerative cells areobtained from one or more different donor subjects, and have beenobtained by obtaining a cardiac biopsy sample from said donor(s), andculturing the sample(s) to obtain the regenerative cells. Suchallogeneic transplant methodology, according to several embodiments, isparticularly beneficial because the regenerative cells do not evoke asignificant chronic immune response that is adverse to the patient.Instead, the regenerative cells trigger a cascade of therapeuticsignaling effects (e.g., a paracrine effect) prior to destruction via anacute immune response that destroys the regenerative cells. Thus,“off-the-shelf” regenerative cells can be produced to treat patientssuffering from cardiac diseases. Further, the patient need not havehealthy tissue from which to harvest his or her own cells (for anautologous transplant). Moreover, even when a patient has viable hearttissue for biopsy, the patient need not have to wait for the culturingprocess when his or her own tissue is not used. Instead, the“off-the-shelf” allogeneic cells may be available with little or no timedelay. In one embodiment, the donor cells are obtained from therecipient patient plus at least one different donor. In one embodiment,the donor cells are obtained from a single donor (different than therecipient patient). In one embodiment, the donor cells are obtained froma two or more different donor (different than the recipient patient andeach other). The recipient patient and donor(s) may be race, age, sex,blood-type, and/or HLA-matched in some embodiments. In otherembodiments, the recipient patient and donor(s) need not be matched inany of the categories identified above. In several embodiments, theallogeneic cells and/or the patient need not be treated with immunesuppressants prior to (or during or after) implantation of theregenerative cells. In some embodiments, the allogeneic cells and/or thepatient need not be treated with radiation prior to (or during or after)implantation of the regenerative cells.

EXAMPLES

Examples provided below are intended to be non-limiting embodiments ofthe invention.

Example 1 Specimen Processing and Cardiosphere Growth

Following institutional guidelines, and with patient consent, humanbiopsy specimens were obtained from patients undergoingclinically-indicated percutaneous endomyocardial biopsy and processed asdescribed above, with certain modifications. Specimens consisted ofwhole or partial bioptome “bites”, stored on ice in high-potassiumcardioplegic solution and processed within two hours (FIG. 1A, step 1).As discussed, herein, samples, in some embodiments, are taken from wholedonor hearts (e.g., not collected via biopsy). Samples were cut intofragments from which gross connective tissue was removed. The fragmentswere then washed, partially-digested enzymatically, and the single cellsdiscarded. In several embodiments, partial digestion is accomplishedusing trypsin. In some embodiments, collagenase is used. In someembodiments, other proteases may be used. The remaining tissue fragmentswere cultured as “explants” on dishes coated with fibronectin (FIG. 1A,step 2). In some embodiments, other surface coatings may be used (e.g.,collagen or other extracellular matrix proteins. After several days, alayer of stromal-like cells arose from adherent explants over whichsmall, round, phase-bright cells migrated. Once confluent, theloosely-adherent cells surrounding the explants were harvested by gentleenzymatic digestion (FIG. 1A, step 3). These cells were seeded at2-3×10⁴ cells/mL on poly-D-lysine-coated dishes in media designed foroptimal growth of cardiospheres (FIG. 1A, step 4). Detachedcardiospheres were then plated on fibronectin-coated flasks and expandedas adherent monolayers (FIG. 1A, step 5), which could be subsequentlypassaged by trypsinization. Single cells were counted under phasemicroscopy using a hemocytometer as cardiosphere-forming cells andduring CDC passaging to track cell growth for each specimen. Isolationof the cardiosphere-forming cells was repeated up to 3 more times fromthe same specimen.

Sub-Population Selection and Flow Cytometric Analysis

To characterize the antigenic features of cells that form cardiospheres,cells obtained during the first harvesting (FIG. 1A, step 3) weresub-selected by magnetic-activated cell separation with anAPC-conjugated monoclonal antibody against c-kit, followed by labelingwith a microbead-conjugated anti-APC, followed by separation usingOctoMACS. CD105⁺ populations were then sub-selected with a secondantibody directly conjugated to a microbead. As discussed herein, inseveral embodiments, selection is used for analysis of the cellpopulation only, and the cells that are administered have not beenselected for expression of a particular marker, including, but notlimited to c-kit.

CDCs were passaged two times as adherent monolayers and then used forflow cytometry experiments. c-kit-APC, CD105-PE, and similarlyconjugated isotype-matched control monoclonal antibodies were utilized.Gates were established by 7-AAD fluorescence and forward scatter. Datawere collected using a FACScalibur cytofluorometer with CellQuestsoftware.

Adenovirus Creation and Cell Transduction

The E. coli beta-galactosidase (lacZ) gene was cloned into an adenoviralshuttle vector pAd-Lox to generate pAd-Lox-LacZ by Cre-Lox recombinationin Cre-4 293HEK cells as described. CDCs were passaged two times andtransduced with virus as adherent monolayers. Transduction efficienciesof 90% were achieved with an MOI of 20 for 12 hours.

Myocardial Infarction and Cell Injection

Adenovirally-transduced CDCs were injected into adult male SCID-beigemice 10-16 weeks of age. Myocardial infarction (MI) was created byligation of the mid-left anterior descending coronary artery and cellsor vehicle injected under direct visualization at two peri-infarctsites. As disclosed herein, other delivery routes (e.g., intracoronary,IV, etc.) are used in some embodiments. CDCs (10⁵) were injected in avolume of 10 μL of PBS (5 μL at each site), with 10⁵ primary human skinfibroblasts or 10 μL of PBS as controls. All mice underwentechocardiography prior to surgery (baseline) and again 20 dayspost-surgery. Ejection fractions (EFs) were calculated using V1.3.8software from 2D long-axis views taken through the infarcted area. Micewere then euthanized at 0, 8, or 20 days, and the excised heartsprepared for histology.

Immunostaining, Immunohistochemistry, and Microscopy

Cardiospheres were collected for immunostaining when they had reached100-1000 cells in size. Primary antibodies against c-kit, CD105, cardiacmyosin heavy chain (cMHC), and cardiac troponin I (cTnI) were used forimmunostaining Secondary antibodies conjugated with Alexa fluorochromeswere utilized. Immunostaining was performed as previously described.Confocal fluorescence imaging was performed on an Eclipse TE2000-Uequipped with a krypton/argon laser using UltraVIEW software.

Mouse hearts were excised, embedded in OCT compound, frozen, andsectioned in 5 μm slices. Tissue sections were stained withhematoxylin-eosin and b-galactosidase reagent or Masson's trichrome.Tissue viability within the infarct zone was calculated from Masson'strichrome stained sections by tracing the infarct borders manually andthen using ImageJ software to calculate the percent of viable myocardiumwithin the overall infarcted area.

Statistics

All results are presented as means±SEM. The significance of differencesbetween any two groups was determined by the Student's t-test. Multiplegroups were compared using GB-Stat software using one-way ANOVA andgroup pairs compared by the Bonferroni-Dunn method if a significant Fvalue was obtained. A value of p<0.05 was considered significant.

The generalized estimation equation (GEE) approach was employed toidentify parameters that were independently associated with high cellyield. Data from patients who donated multiple specimens were treated asrepeated measures. Those parameters that were significant (p<0.1) in theunivariate models were included in the final, multivariate models. Theanalysis was performed with the use of SAS software. A final value ofp<0.05 was considered significant. All p-values reported are 2-sided.

Statistics

Product manufacturers, recipes, and reagents used in several embodimentsare shown in Table 1.

TABLE 1 Products and Manufacturers and Media Recipes Explant and CDCmedia IMDM 20% FBS  1% penicillin-streptomycin  1% L-glutamine 0.1 mM2-mercaptoethanol Cardiosphere media  35% IMDM and 65% DMEM/F-12 Mix3.5% FBS   1% penicillin-streptomycin   1% L-glutamine 0.1 mM2-mercaptoethanol Thrombin, B-27, bFGF, EGF and Cardiotrophin-1 at finalworking concentrations Product: Working concentration: Manufacturer:IMFM Invitrogen DMEM/F-12 Mix Invitrogen Thrombin  1 unit/mL Sigma B-271:50 Invitrogen bFGF 80 ng/mL PeproTech EGF 25 ng/mL PeproTechCardiotrophin-1  4 ng/mL PeproTech Fibronectin 25 μg/mL BD BiosciencesPoly-D-lysine 20 μg/mL BD Biosciences c-kit-APC 1:10 BD Pharmingen CD105MicroBeads 1:5 Miltenyi Biotec Anti-APC MultiSort 1:4 Miltenyi BiotecCD105-PE 1:10 R&D Systems 7-AAD 20 μg/mL Calbiochem SCID-beige miceHarlan Dermal fibroblasts ATCC c-kit pAb 1:100 Abcam CD105 mAb 1:50 R&DSystems cMHCpAb 1:100 (6) Rome, Italy cTaImAb 1:200 Chemicon Alexa 488,568 1:400 Invitrogen OCT VWR Scientific Equipment and Software:Manufacturer: OctoMACS Miltenyi Biotec FACScalibur BD Biosciences Vevo660 Echo VisualSonics Eclipse TE2000-U Nikon CellQuest BD BiosciencesV.1.3.8 software VisualSonics UltraVIEW software Perkin Elmer ImageJsoftware NIH GB-Stat V10 Dynamic Microsystems Inc. SAS software v9.1 SASInstitute Inc.

Example 2 Specimen Processing and Cardiosphere-Forming Sub-Populations

FIG. 1B shows a typical explant, after mincing and partial enzymaticdigestion, on the day it was obtained and also on days 3 (FIG. 1C) and13 (FIG. 1D), immediately prior to first harvest. Harvesting ofcardiosphere-forming cells (FIG. 1A, step 3) was initially performed 8or more days after obtaining a specimen and at 4-12 day intervalsthereafter. Panel E summarizes the results of sub-population selectionexperiments performed using cells harvested from 3 different patientspecimens. The large majority of the cells that generate cardiospheresare CD105⁺, those that are c-kit⁺ and those that are c-kit⁻. Typicalcardiospheres are shown in FIG. 1F, 12 days after harvest. Floatingcardiospheres were plated for expansion (FIG. 1A, step 5) 4-28 daysafter step 3 and passaged at 2-7 day intervals thereafter. FIG. 1G showsCDCs plated on fibronectin during expansion at passage 2, when thosecells were harvested for injection.

Example 3 Patient Specimens and Cardiosphere Growth

83 patient specimens (21.0±1.9 mg) were obtained for analysis. 72 of thespecimens were obtained from patients who had received a hearttransplant and 11 were from patients awaiting transplant. Ninetransplanted patients donated multiple specimens. 78 of 83 specimenswere processed, and 4 of those specimens never harvested were fromrepeat patients, yielding growth data from 69 of 70 patients. Cumulativegrowth curves for each specimen are depicted in FIG. 1, Panels H and I.The growth curves from patients awaiting transplant (FIG. 1H) aresimilar to those from transplanted patients (FIG. 1I), showing a widerange of growth potential among specimens. Patient parameters aresummarized in Table 2 for the non-transplanted and transplanted groups.A GEE analysis involving all patient parameters listed in the tablerevealed no independent predictors for high cell yield within thenon-transplanted group. Within the transplanted group, specimens frompatients with a higher EF tended to yield more cells, but the effect wasweak (final R² estimate=0.04, p<0.05).

TABLE 2 Patient Population Summary Non-transplanted Patients:Transplanted Patients: Patient age 47.2 ± 3.7 years 53.6 ± 1.7 yearsPatient sex 63% male, 37% female 73% male, 27% female Patient ejection36.9 ± 4.7% 61.9 = 0.8% fraction Donor age 31.4 ± 1.6 years Donor sex69% male, 31% female Time out from  4.5 ± 0.6 years transplant Donorischemic 173.9 ± 7.8 minutes time Pathological 0.5 ± 0.1 rejectionlevel* Immunosupportive 31% normal, 43% low, level** 26% high *grade 0 =0, grade 1A = 0.5, grade 1B = 1, grade 2 = 2, grade 3A = 3 **consideredfor Cyclosporine and FK506 (±Rapamycin) relative to time out fromtransplant.

Example 4 Cardiosphere and Cardiosphere-Derived Cell Phenotypes

Part of the rationale for using CDCs lies in the unique biology ofcardiospheres and their cell progeny. The self-organizing cardiospherescreate a niche environment favoring the expression of stem cell antigens(e.g., c-kit and CD105, FIG. 2A) and frequently manifest a surfacephenotype marked by mature cardiac-specific antigens (cMHC and cTnI,FIG. 2B) with retention of internal “stemness”. In fact, c-kit and CD105were present in all cardiospheres examined (10 or more from each of 10patients), with c-kit either localized to the core or expressedthroughout the sphere, and CD105 typically localized to the periphery orexpressed throughout. CDCs after two passages retain high levels ofc-kit and CD105 antigen expression (FIG. 2C, representative ofexpression profiles of CDCs from 3 and 2 different patientsrespectively).

Example 5 Cardiosphere-Derived Cell Engraftment, Regeneration, andFunctional Improvement

CDCs from 4 different patients were utilized for in vivo experiments. Toassess engraftment and cell migration, mice were injected withlac-Z-expressing CDCs and sacrificed at each of 3 time points (0, 8, and20 days following injection). At day O, CDCs were located at injectionsites in the border zone, but at day 8 and day 20 injected cells weredistributed mainly within the MI area, forming islands or continuousbands of β-galactosidase positive tissue (see e.g., FIG. 5).

Eight mice were injected with CDCs and followed for 20 days; 11 miceserved as controls (4 with fibroblasts, and 7 with PBS). FIG. 3A shows atypical beta-galactosidase staining pattern indicating the distributionof injected human cells after 20 days in vivo. Note the band of bluecells infiltrating the infarct zone, which was not apparent in thefibroblast-injected mice (FIG. 3B) or the PBS-injected mice. Masson'strichrome-stained sections were used to quantify regeneration (FIG. 3, Cand D). Panel C, from a CDC-injected heart, shows a number of obviousred regions within the blue infarct zone; fewer such regions are evidentin the fibroblast-injected heart (FIG. 3D). CDC-injected mice had ahigher fraction of viable fuchsin-positive tissue within the MI zone(24.9±1.1%) compared to fibroblast-injected mice (17.7±1.8%, p<0.01) orPBS-injected mice (13.7±0.7%, p<0.01), but the overall total infarctarea was similar to that in the two control groups (60.6±6.4 CDC,76.9±7.0 fibroblast, 75.7±2.7 PBS, units in 10⁴ pixels; p=NS). Thedifferences between the CDC group and each of the control groups inpercent viable myocardium within the MI zone, 7.2% and 11.2%, representthe extents of myocardial regeneration attributable to the CDCs.

Echocardiograms were performed for all groups at 20 days; FIG. 4 showsexamples from the CDC and fibroblast-treated groups at end-diastole andend-systole. Pooled data for left ventricular EF (LVEF, FIG. 4E) andleft ventricular fractional area (LVFA, FIG. 4F) reveal a higher LVEF inthe CDC-treated group (38.8±1.7%) as compared to either thefibroblast-treated (24.5±1.8%, p<0.01) or the PBS-treated group(26.4±3.0%, p<0.01), but the two control groups were indistinguishable.There was no difference among the LVEFs at baseline.

Example 6 Process for the Isolation of Cardiac Stem Cells from CardiacBiopsy Specimens

Pluripotent stem cells may be isolated from cardiac biopsy specimens orother cardiac tissue using a multi-step process (see FIG. 1A forschematic). First, cardiac tissue is obtained via percutaneousendomyocardial biopsy or via sterile dissection of the heart. Onceobtained, tissue specimens are stored on ice in a high-potassiumcardioplegic solution (containing 5% dextrose, 68.6 mmol/L mannitol,12.5 meq potassium chloride, and 12.5 meq sodium bicarbonate, with theaddition of 10 units/mL of heparin) until they are processed (up to 12hours later). For processing, specimens are cut into 1-2 mm.sup.3 piecesusing sterile forceps and scissors; any gross connective tissue isremoved. The fragments are then washed with Ca⁺⁺Mg⁺⁺ free phosphatebuffered saline (PBS) and typically digested for 5 min at roomtemperature with 0.05% trypsin-EDTA. Alternatively the tissue fragmentsmay be digested in type IV collagenase (1 mg/mL) for 30 minutes at 37degrees C. Preliminary experiments have shown that cellular yield isgreater per mg of explant tissue when collagenase is used.

Once digestion is complete, the remaining tissue fragments are washedwith “Complete Explant Medium” (CEM) containing 20% heat-inactivatedfetal calf serum, 100 Units/mL penicillin G, 100 μg/mL streptomycin, 2mmol/L L-glutamine, and 0.1 mmol/L 2-mercaptoethanol in Iscove'smodified Dulbecco medium to quench the digestion process. The tissuefragments are minced again with sterile forceps and scissors and thentransferred to fibronectin-coated (25 μg/mL for at least 1 hour) tissueculture plates, where they are placed, evenly spaced, across the surfaceof the plate. A minimal amount of CEM is added to the plate, after whichit is incubated at 37° C. and 5% CO₂ for 30 minutes to allow the tissuefragments, now referred to as “explants”, to attach to the plate (FIG.1B). Once the explants have attached, enough CEM is added to the plateto cover the explants, and the plates are returned to the incubator.

After a period of 8 or more days, a layer of stromal-like cells beginsto arise from adherent explants, covering the surface of the platesurrounding the explant. Over this layer a population of small, round,phase-bright cells is seen (FIG. 1C, 1D). Once the stromal cell layerbecomes confluent and there is a large population of bright phase cells,the loosely-adherent cells surrounding the explants are harvested. Thisis performed by first washing the plate with Ca⁺⁺Mg⁺⁺-free PBS, thenwith 0.48 mmol/L EDTA (for 1-2 min) and finally with 0.05% trypsin-EDTA(for 2-3 min). All washes are performed at room temperature under visualcontrol to determine when the loosely adherent cells have becomedetached. After each step the wash fluid is collected and pooled withthat from the other steps. After the final wash, the explants arecovered again with CEM and returned to the incubator. Each plate ofexplants may be harvested in this manner for up to four times at 5-10day intervals. The pooled wash fluid is then centrifuged at 1000 rpm for6-8 minutes, forming a cellular pellet. When centrifugation is complete,the supernatant is removed, the pellet is resuspended, and the cells arecounted using a hemacytometer. The cells are then plated inpoly-d-lysine coated 24-well tissue culture plates at a density rangingfrom 3-5×10⁴ cells/well (depending on the species) and returned to theincubator. The cells may be grown in either “Cardiosphere Growth Media”(CGM) consisting of 65% Dulbeco's Modified Eagle Media 1:1 with Ham'sF-12 supplement and 35% CEM with 2% B27, 25 ng/mL epidermal growthfactor, 80 ng/mL basic fibroblast growth factor, 4 ng/mL Cardiotrophin-1and 1 Unit/mL thrombin, or in CEM alone.

In either media, after a period of 4-28 days, multicellular clusters(“cardiospheres”) will form, detach from the tissue culture surface andbegin to grow in suspension (FIG. 1E, 1F). When sufficient in size andnumber, these free-floating cardiospheres are then harvested byaspiration of their media, and the resulting suspension is transferredto fibronectin-coated tissue culture flasks in CEM (cells remainingadherent to the poly-D-lysine-coated dishes are not expanded further).In the presence of fibronectin, cardiospheres attach and form adherentmonolayers of CDCs (FIG. 1G). These cells will grow to confluence andthen may be repeatedly passaged and expanded as CDCs, or returned topoly-d-lysine coated plates, where they will again form cardiospheres.Grown as CDCs, millions of cells can be grown within 4-6 weeks of thetime cardiac tissue is obtained, whether the origin of the tissue ishuman (FIG. 1I), porcine or from rodents (data not shown). Whencollagenase is used, the initial increase in cells harvested per mass ofexplant tissue results in faster production of large numbers of CDCs.

Example 7 Evaluation of Processing and Culture Conditions

Optionally, changes in processing or culture methods and or reagentsdisclosed herein may be made in order to promote generation ofcardiospheres, CDCs, or IICSps in sufficient numbers and havingsufficient viability for use in allogeneic (or autologous) therapies

In several embodiments a commercially-available cardioplegic solution isused to store the biopsy or donor tissue until processing begins. Insome embodiments, the initial biopsy digestion step is modified throughthe used of with collagenase rather than trypsin. In some embodiments,the number of mincing steps is minimized.

In several embodiments the presence of recombinant human proteins and/orcytokines in cardiosphere medium are eliminated, and fetal bovine serumused in their place. However, in some embodiments, recombinant humanproteins and/or cytokines are used in combination with fetal bovineserum. In several embodiments, gentamicin is used as an antibiotic. Insome embodiments, gentamicin is preferred as compared to penicillin andstreptomycin, or combinations thereof. In several embodiments,antibiotics are removed from the culture media in the final phase of CDCculture. In several embodiments, trypsin is replaced with TrypLE Selectduring the harvesting of cardiosphere-forming cells and passaging ofCDCs.

Tissue collected sites were also compared for CDC yield. The sites werethe right ventricular septal wall (RVS), the atria, the apex, the rightventricular epicardium (RVE), and the left ventricular epicardium (LVE).As shown in FIG. 7A, yields from the atria and RVE are somewhat greaterthan yields from the other regions. Thus in some embodiments these sitesare preferred (whether from biopsy or whole heart). However, these dataalso show that CDCs can reliably be produced using tissue from each ofthe collection regions tested. In some embodiments, other regions of theheart are used.

Because several embodiments involve the use of a heart that is incondition for transplant, there may be a delay from the collection ofthe heart itself and the initiation of processing the heart to generateCDCs. Therefore, the effect of tissue storage on CDC yield was examined.Specimens were taken from two transplant quality human hearts. Specimensprocessed immediately after collection were compared to those stored for3 or 6 days in cold cardioplegia solution or to those cryopreserved andsubsequently thawed. For each of the two hearts, 6-12 specimens wereprocessed following each storage condition. Four out of 12 specimenstaken from the first heart and stored for 6 days did not yield CDCs, andtherefore, in some embodiments, tissue is preferably stored for lessthan 6 days (e.g., 1, 2, 3, 4, or 5 days). However, in severalembodiments, storage for 6 days is acceptable, as the possibility ofgenerating CDCs from such a sample exists.

Data demonstrate a slight effect of 3 days of cold storage in terms ofthe time required to achieve a similar CDC yield, and a larger effectseen after freezing and thawing the specimen (FIG. 7B). Freezing andthawing decreased the average yield and increased processing timerequired. However, following either 3 days of cold storage orcryopreservation CDCs were reliably generated. Thus in severalembodiments, tissue is processed for CDCs immediately, while in someembodiments, a storage time of 1-2 or 2-3 days (or longer) is allowed.In some embodiments, cold storage is used, while in other embodiments,cryopreservation is used.

Tissue samples from the RVS and cultured in conditions such as thosedescribed above are used, in some embodiments, to generate a master cellbank (MCB). CDCs were passaged to P1 to create the MCB. A fraction ofCDCs underwent further passaging to P6 in order to generate a workingcell bank (WCB). The yields presented for the WCB (Table 3) representthe potential yield extrapolated from the growth seen with the fractionof cells expanded. In some embodiments, greater passage numbers are usedfor either the MCB or the WCB. For example, the MCB is optionallygenerated at P2, P3, P4, P5, or P6. Likewise, the WCB is optionallygenerated at P7, P8, P9, P10, P11, P12, or more. In some embodiments,CDCs are expanded just prior to the point of to the point of in vitrosenescence). The WCB is then split into multiple doses, e.g., singlecryopreserved doses.

These data taken as a whole, demonstrate the feasibility of generatingCDCs and cell banks from the CDCs from transplant-quality hearts.

TABLE 3 Cell Banks Generated from Whole Hearts Source MCB Yield WCBYield Donor Heart 1 116 million 8.72 billion Donor Heart 2 185 million42.0 billion

Example 8 Characterization of Human CDCs

The markers expressed on CDCs, in several embodiments, are related tothe “sternness” of the cells. In some embodiments, markers are used toselect, screen, isolate, or enrich a sample for cells bearing one ormore markers. In some embodiments, however, cells are isolated withoutpreference to a given marker or set of markers. The vast majority ofCDCs (derived from 13-27 different preclinical patient endomyocardialbiopsy specimens) were CD105⁺, with significant pluralities that wereCD90⁺ and c-kit⁺ (FIG. 8A). CDCs were largely negative for CD45(0.1±0.1%). FIG. 8B demonstrates that CDCs contain distinctsub-populations of cells that are CD105⁺CD90⁺c-kit⁻. These particularprofiles, in some embodiments are suggestive of cardiac mesenchymalcells or fibroblasts. CDCs also, in some embodiments, contain distinctcardiac progenitor populations (c-kit⁺CD90). In several embodiments, allpopulations express CD105, the regulatory component of the TGF-βreceptor complex important in angiogenesis and hematopoiesis. In severalembodiments all populations also lack CD45. In some embodiments, CD45 isused to screen for contaminating blood-derived cells to ensure purity ofthe CDCs to be administered.

Data presented in Example 10 illustrate the potency of thesub-populations in comparison to the total population in an animalmodel.

Example 9 In Vitro Differentiation of Human CDCs CardiomvocvteDifferentiation

In order to examine the ability of human CDCs to differentiate fullyinto functional cardiomyocytes, an in vitro co-culture system wasutilized. DiI-labeled or lentivirally-transduced GFP⁺ CDCs wereidentified in co-cultures with neonatal rat ventricular myocytes (NRVMs)which spontaneously contract in culture. Cocultures were subjected toimmunostaining or whole-cell patch clamp in order to recordvoltage-sensitive currents. Co-cultured CDCs demonstrated biophysicalfeatures characteristic of cardiomyocytes, including: contractions asearly as 24 hours after the start of co-culture, sarcomeric organization(FIG. 9A), expression of the inwardly-rectifying potassium currentI_(K1) which is consistent with a cardiomyocyte ventricular phenotype(FIG. 9B), and when transduced with the β-subunit of the L-type calciumchannel expression of the calcium current I_(Ca,L) indicating thepresence of the pore-forming α-subunit (FIG. 9C). Thus, in severalembodiments, CDCs differentiate into cardiomyocytes having normalfunctional characteristics. Therefore, such CDCs, upon administration,are used in some embodiments to effectuate cardiac repair.

Endothelial Differentiation

Human CDCs were also challenged to an endothelial tube-forming assay byculturing them on MATRIGEL™ in endothelial differentiation media. Within4-6 hours, human CDCs formed complex tube networks (FIG. 10A) resemblingthose created by human umbilical vein endothelial cells (HUVECs) (FIG.10B). Unexpectedly, CDCs display a unique morphology between about 24and about 72 hours after the start of the assay. CDCs contract towardone another within about 24 hours and then begin to migrate into theunderlying gel substrate within 72 hours. This response could bereversibly inhibited by including HERCEPTIN® in the endothelial media(data not shown), a factor known to inhibit angiogenesis. Thus, inseveral embodiments, CDCs are capable of forming endothelial cells, andin some embodiments, such endothelial cells are related to angiogenesis.These data demonstrate the potential for administered CDCs to generatenot only cardiomyocytes, but also to generate supporting endothelialcells and, in some embodiments, new vasculature to supply newlygenerated cardiomyocytes with blood.

Example 10 Mouse Model of Allogeneic Cardiac Repair Protocols forTesting Human Cdcs in Mouse Model

As a supplement to the Examples described above, myocardial infarctionwas created in adult male SCID-beige mice by permanent ligation of theleft anterior descending (LAD) coronary artery, as described above.Cells were delivered intramyocardially by direct injection at twoperi-infarct sites immediately following ligation. CDCs (10⁵) wereinjected in calcium-free PBS (5-7 μL at each site), with 10⁵ normalhuman dermal fibroblasts (NHDFs) or PBS as controls. During the courseof the study, mice underwent echocardiography prior to surgery(baseline), and at 2 days, 3 weeks, and 6 weeks post-surgery. Leftventricular ejection fraction was evaluated by manual planimetry of theendocardial border in end-diastolic and end-systolic frames. Mice wereeuthanized at the end of the study for histology. Human cells wereidentified in histological sections using a human-specific monoclonalantibody and a human-specific DNA probe.

Time Course of Engraftment of Human Cdcs in Infarcted Mice

In order to track the time course of migration of human CDCs injectedinto infarcted mice, an in vivo bioluminescence study was performed,with treated animals being sacrificed at various time points forhistology. To investigate acute cell retention in the heart and tovisualize the biodistribution of the cells over the short term, CDCswere transduced with a lentivirus containing the luciferase gene.Animals were given an injection of D-luciferin intraperotineally andsubjected to optical imaging on an IVIS® SPECTRUM (Xenogen) 1 day, 4days, and 1 week after cell delivery. Images were acquired every 4minutes with a 1 minute exposure time until the peak signal was obtainedand the signal began to decline. FIG. 11A depicts a strong luminescentsignal present in the heart out to 1 week in a representative animal(n=3). Cells were not detectable in other organs. This approachdemonstrates acute survival and localization of the delivered cells.Peak luminescence values normalized to the day 1 value detected in thehearts of each animal are shown in the graph in FIG. 11B.

Regardless of the apparent decrease in the number of detectable cells byabout 1 week, overall morphological and functional improvement in theCDC-treated group persists for at least 6 weeks post-Ml (see e.g., FIGS.12A-12K and FIG. 13). These data suggest that the therapeutic effects ofthe CDCs are initiated at an early post-administration time point. Thus,these data support the concept of a persistent effect of theadministered cells that is not dependent on the continued presence ofviable cells and perhaps recruits endogenous signaling cascades and/orendogenous cells to maintain the effect. To that end, in severalembodiments, transient engraftment of the cells into recipient cardiactissue is sufficient to yield anatomical and/or functional benefits.

Histologically, human cells were detected by immunostaining using anantibody against a human nuclear antigen (shown below as greenfluorescent nuclei among the blue fluorescence of all nuclei). Arepresentative Masson's Trichrome stained section shows the extent ofthe infarct at each timepoint examined (FIG. 12A-C). Low magnificationimages demonstrate the presence of human CDCs in the infarct border zoneinjection site at 2 days post-MI (FIGS. 12D and 12E are from the boxedarea in FIG. 12A) and throughout the border zone and infarct itself at 1week post-MI (FIGS. 12F and 12G are from the boxed area in FIG. 12B).Over the course of the 6 week study period, CDCs distributed throughoutthe infarct, border zone, and eventually the remote myocardium (FIGS.12H-12K are from the boxed areas in FIG. 12C). The majority of CDCscould be found engrafted throughout the infarct region (57±3% of thetotal engrafted) and the immediate border zone (30±5%), but stableengraftment also existed in the remote myocardium (13±3%). Engraftmentof CDCs into murine cardiac tissue was also confirmed by western blotusing human-specific antibodies (see FIG. 22). Human CDCs reconstitutedlarge portions of the mouse hearts 6 weeks after delivery.

These data therefore demonstrate that, in several embodiments, CDCsadministered to a subject having damaged or diseased cardiac tissue arecapable of being retained in damaged or diseased cardiac tissue for bothshort term and longer time frames. In some embodiments, cells areretained for between about 1-10 days, 2-9 days, 3-8 days, or 4, 5, or 6days, or overlapping ranges thereof. In some embodiments, there isdetectable cell loss (e.g., loss of retention of the cells in the tissueand/or loss of viability of the cells) over the short term. In someembodiments, no or limited cell loss occurs, and administer cells areretained for about 1 week, about 4 weeks, about 6 weeks, or longer. Insome embodiments, cells are retained for between about 1-2 weeks, 2-3weeks, 3-4 weeks, 4-6 weeks, 6-10 weeks, 10-15 weeks, and overlappingranges thereof. In some embodiments, cells are permanently retained(e.g., for the lifetime of the recipient). As discussed more fullybelow, in some embodiments, the long-term retention of cells within atarget site is not critical to the successful regeneration of cardiactissue (or improvement in cardiac function).

Efficacy of Human CDCs in Mouse Model

To assess efficacy, CDCs from 21 different randomly-selected preclinicalpatients were utilized for functional experiments. Twenty-one mice weresubject to experimental MI (as described above) were injected with CDCs.Thirty-five mice served as controls (17 injected with normal humanneonatal dermal fibroblasts (NHDFs), and 18 with PBS). Echocardiogramsperformed in normal mice prior to MI revealed a left ventricularejection fraction (LVEF) of ˜80%. Two days after MI, there were nosignificant differences among the groups in terms of LVEF, although allhad a substantial decline in function indicating a successful MI thatwas uniform in severity in all groups. Data for all animals summarizedin FIG. 13. CDC-injected animals showed no significant deterioration ofLV function from 2 days post-MI to 3 weeks. Moreover, CDC— injectedanimals showed no decrease in function at 6 weeks post-MI. Given therelatively long-term time point for a mouse model (>10% of the typicalmurine lifespan of ˜1 year), persistence CDC of the benefit wasunexpected. At 3 and 6 weeks, LVEF was significantly higher in theCDC-treated group than in either the NHDF treated or the PBS-treatedgroup. LVEF in the two control groups were indistinguishable from eachother.

In several embodiments, administration of CDCs prevents further loss ofcardiac function due to MI. In some embodiments, loss of cardiacfunction is less than about 45% to about 40%, less than about 40% toabout 35%, less than about 35% to about 30%, less than about 30% toabout 25%, less than about 25% to about 20%, less than about 20% toabout 15%, less than about 15% to about 10%, less than about 10% toabout 5%, less than about 5% to about 1%, and overlapping rangesthereof. In some embodiments, loss of function is reduced compared tonon-CDC treatments. In some embodiments, cardiac function is increasedover time, based on the administration of CDCs.

In several embodiments, administration of CDCs induces increases incardiac function due post-MI. In some embodiments, cardiac function isincreased by up to about 5%, up to about 10%, up to about 15%, up toabout 20%, up to about 25%, up to about 30%, up to about 35%, up toabout 40%, and overlapping ranges thereof. In some embodiments, cardiacfunction is increased to levels beyond those levels existing pre-MI.

In several embodiments, CDC administration provides an initial shortterm benefit. In some embodiments, this initial benefit continues for alonger time period. In some embodiments, the time frame ranges fromabout 3-6 weeks, or longer. In several embodiments, the benefit isrealized for about 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-6 weeks, 6-10weeks, 10-15 weeks, and overlapping ranges thereof. In some embodiments,the CDC-derived benefit is realized for the lifetime of the recipient.

A morphometric analysis was also conducted at the 6 week study endpointto assess the effects of CDC treatment on infarct remodeling. Mousehearts were excised, washed in PBS, arrested in diastole with ice-coldKCl, frozen and sectioned transversely in 5-8 μm slices. For amorphometric analysis, tissue sections were selected from the largestextent of the infarct area and stained using Masson's Trichrome(representative results are shown in FIG. 12). Photographs encompassingthe entire section were acquired. Infarct wall thicknesses and tissueviability within the infarct zone were calculated from Masson'sTrichrome-stained sections by tracing borders manually and then usingImageJ software (NIH) to make measurements. Three to six sections wereanalyzed per animal and values averaged. In several embodiments,administration of CDCs results in a larger tissue thickness in theinfarct area. In turn, in some embodiments, the larger thickness resultsin an increased percentage of viable myocardium. In several embodiments,percentage of viable myocardium in the infarct area ranges from about15% to about 20%, from about 20% to about 25%, from about 25% to about30%, from about 30% to about 35%, from about 35% to about 40%, andoverlapping ranges thereof. In some embodiments, viable myocardium inthe infarct zone is greater than 40%. In some embodiments, theseincreases are also associated with increased cardiac function.

In addition, whole hearts and Masson's Trichrome-stained tissue sectionswere examined for evidence of tumor formation. No tumors were detectedin the hearts of any animal. Thus, in several embodiments allogeneicadministration of CDCs serves accomplish one or more of increasingcardiac function, increasing cardiac tissue thickness in the infarctzone, increase the percentage of viable myocardium in the infarct zone.In some embodiments, the above are accomplished in the absence of tumor(e.g., teratoma) formation.

Efficacy of CDC Sub populations in Mouse Model

The efficacy of the cardiac mesenchymal cell and the cardiac progenitorcell sub-populations alone were tested in comparison to the total CDCpopulation. A magnetic-activated cell sorting technique was utilized toenrich the sub-populations of interest, CD90⁺ cardiac mesenchymal cellsand c-kit⁺ cardiac progenitor cells. CDCs were sorted using theCELLection Pan Mouse IgG Kit (Invitrogen) and a Dynal Magnetic ParticleConcentrator-15 (Invitrogen). The following monoclonal antibodies wereutilized for the first labeling step: CD90-FITC (1:10, Dianova),c-kit-APC (1:10, BD Pharmingen), and CD105-PE (1:10, R&D Systems). Afterstaining and washing, cells were labeled with prewashed CELLectionDynabeads conjugated via a DNA linker to a secondary antimouse IgGantibody. After staining and diluting, the labeled cell solution wasplaced into the MPC-15 and the unlabeled cell fraction aspirated anddiscarded. The labeled cell fraction was resuspended in releasing bufferto allow for cleavage of the DNA linker and release of the Dynabeadsfrom the cells. The cell and Dynabead solution was placed into theMPC-15, the cell fraction was collected and the Dynabeads werediscarded. After another wash, cells were resuspended in media forculture overnight (prior to in vivo delivery).

Enriched CDC sub-populations were then tested in the same mouse MI modeldescribed above. CDCs enriched for CD105, present on 97% of all CDCs,were used as a control. The cardiac function (as measured by LVEF) ofmice with a CD-105-enriched population was comparable to CDC-injectedmice. See FIG. 14. These data indicate that the sorting protocol did notitself significantly impair the therapeutic potential of CDCs. Thus, inseveral embodiments, cell sorting for one or more particular markerspresent on (or absent from) CDCs is used to enrich a population of CDCsfor one or more particular markers.

LVEF for c-kit- and CD90-injected mice were indistinguishable from oneanother. Both of these groups were significantly outperformed by theCD105-injected mice and the CDC-injected mice. All groups were thencompared to mice treated with fibroblasts and mice treated with PBS.c-kit-injected mice had significantly greater LVEF than both fibroblast-and PBS-injected mice. The CD90-injected group approached significancewhen compared with the fibroblast and PBS groups. While the therapeuticmechanisms of action of these two distinct sub-populations may differ,both offer similar global functional benefits to those enriched forCD-105 and unenriched CDCs. Thus, in some embodiments, cellsorting/enrichment is used to prepare a sub-population for use inallogeneic therapies. However, in several embodiments, use of a CDCpopulation that has not been enriched for any one marker (including butnot limited to c-kit) is particularly advantageous, as the requiredmanipulation and handling of the cells is reduced. Thus, the generationof a population of CDCs for allogeneic therapies is simpler, more rapid,and less likely to be affected by contamination.

Differentiation of Human CDCs in Infarcted Mice

The extent to which CDCs proliferated and formed new cardiomyocytes overthe study time period (e.g., the 6 weeks post-MI) was investigated.Mitotically-active CDCs were identified by expression of ahuman-reactive Ki67 (FIG. 15A-15C). The percentage of Ki67⁺ CDCsincreased from 2 days to 4 days post-MI from 4% to 6% (7 of 112counted), after which time it became difficult to detect proliferativeCDCs (see FIG. 15D-15F). As many as 25% (28 of 111 counted) of CDCs werecardiac-committed at 2 days post-MI as evidenced by expression of Nkx2.5(FIG. 15G-15I). Human CDCs did not express cTnI at 2 days post-MI (FIG.15M-15O), but could be found lodged within regions of dead and dyingcardiomyocytes (evidenced by loss of sarcomeric organization andanucleation). By 1 week post-MI, infiltration of mouse progenitors intothe infarct was apparent. Clusters of Nkx2.5⁺ cells of both human(yellow arrows) and mouse (white arrows) origin were prominent. Afterthe early proliferative CDC response had largely resolved, cardiomyocytedifferentiation appeared to commence, as newly forming cardiomyocytes ofboth human and mouse origin could be identified within the infarct.These newly forming cardiomyocytes were identified as such due diffusecytoplasmic expression of cTnI (see FIG. 15P-15R).

At the end of the 6 week period, CDCs had formed not onlycardiomyocytes, but also non-cardiomyocytes throughout the heart, asdemonstrated in FIG. 16. FIG. 16 shows fluorescence in situhybridization using a human-specific centromeric probe and the redfluorescence of cardiac troponin I (FIGS. 16A-16D). Human nuclei ofinterest are outlined in FIGS. 16E-16H. Example cardiomyocyte nuclei areshown in FIGS. 16M-16P at higher magnification.

These data indicate that, in several embodiments, CDCs not only have thecapacity to proliferate, but CDCs also have the capacity todifferentiate and repopulate damaged myocardium by formingcardiomyocytes as well as non-cardiomyocytes. Moreover, in severalembodiments, administered CDCs, also attract endogenous cardiacprogenitor cells. Taken together, in several embodiments, one or more ofthese characteristics of CDCs are responsible for the resultant increasein cardiac tissue viability, regeneration, and/or overall function.

In Vitro Analysis of Paracrine Factors from Regenerative Cells

In order to characterize the paracrine signals that, in severalembodiments provide a beneficial therapeutic effect, the cytokines andgrowth factors released from regenerative cells were screened. Alsoassessed was whether the released cytokines and growth factors yieldfavorable biological effects on neonatal rat ventricular myocytes(NRVMs) and human umbilical vein endothelial cells (HUVECs).

Human cardiospheres and CDCs were obtained from percutaneous septalendomyocardial biopsies from 21 different patients as described above.

Media were conditioned for 48 hours by cardiospheres and IICSps after4-5 days of culture on poly-D-Lysine, or by CDCs and NHDFs when theywere approximately 90% confluent. Media for conditioning comprised 2.5%FBS complete explant medium (CEM), or glucose-free FBS-free basal medium(BM): Medium 199, 10 mmol/L HEPES, 0.1 mmol/L MEM non-essential aminoacids, 2 mmol/L L-glutamine, 0.8 μg/mL vitamin B12, 2 unit/mLpenicillin. Conditioned media (CM) were stored at −80° C. until used. Insome experiments, CMs were pre-incubated on a shaker for 1 hour at roomtemperature with anti-VEGF and/or anti-HGF neutralizing antibodies. Asdiscussed above, changes to the culturing protocol (e.g., % oxygen,media changes, and the like) are used in some embodiments.

Serum-free CMs from and CDCs were screened for secreted factors using aprotein array according to the manufacturer's protocol (Ray Biotech).

Neonatal rat ventricular myocytes (NRVM) were isolated by standardprocedures known in the art. Culture plates were incubated in humidified2% O₂ atmosphere for 24 or 72 hours with CM or FBS-free BM (lowerportion of FIG. 19). A portion of the cells were then collected bytrypsinization, labeled with Annexin V-FITC and 7AAD and analyzed byflow cytometry for indications of apoptosis.

Human umbilical vascular endothelial cells (HUVEC) were plated onpre-cast, matrix-coated 96-well plates. They were plated with eitherendothelial cell media (ECM), as a positive control, or with CM, or withFBS-free BM, as the negative control (see FIG. 17 for a protocoloutline). After 18 hours, total tube length formed was measuredmicroscopically.

Cell cultures were lysed in lysis buffer (20 mmol/L TrisHCl, 5 mmol/LEDTA, 50 mmol/L NaCl, 1% SDS) with proteinase inhibitors cocktail(Sigma), and homogenized by sonication. Tissue samples were lysed inlysis buffer with proteinase inhibitors cocktail (Roche) and homogenizedwith a rotor-stator homogenizer. Homogenates were spun at 12,000 rcf for15 minutes at 4° C. Supernatants were then collected and stored at −80°C., after quantification by Lowry assay of the protein content (BioRad).Western blots were performed as described above. Primary antibodies usedwere as follows: hVEGF and pan-GAPDH (Abcam), hHGF and hIGFI (R&DSystems), hGAPDH (LabFrontier), Akt (Cell Signaling Technology), Caspase3 (Csp3; Santa Cruz). Human specificity was confirmed. Membranes werewashed in TBST, incubated with HRP-conjugated secondary antibodies(Pierce; Santa Cruz), and developed with ECL (Amersham) or West-Femtosubstrate (pierce). Western blots on media were performed with theNupage system, loading 30 μl of media per lane. After blocking,membranes were incubated with HRP-conjugated anti-mouse IgG antibody(Santa Cruz). Densitometric analysis was performed with ImageJ softwareand plotted as ratios to the GAPDH signal.

RNA from cells and tissue samples was extracted with column-based kits(Qiagen). Reverse transcription was performed on lug starting RNA(Stratagene) in a 20 μl reaction, and 2 μl of cDNA product were thensubjected to PCR (Invitrogen) with human specific or pan-specificprimers for 35 thermal cycles.

In preparation for immunofluorescence and histology, cardiospheres andCDCs were fixed for 10 minutes with ethanol-acetone 50:50% at 4° C.Hearts were cut in 5 μm sections. After deparaffinization andrehydration of the tissue sections, slides were washed and permeabilizedwith 0.1% Triton X-100 in PBS with 1% BSA, then blocked in 10% goatserum and incubated overnight at 4° C. in 1% goat serum with primaryantibodies: anti h VEGF and human nuclear antigen (HNA; Chemicon), hHGF,hIGFI and CD 105 (R&D Systems), FN, KDR and c-kit (Abeam), Met andnk×2.5 (Santa Cruz), IGFI-R (Upstate). Slides were then washed andincubated with Alexa Fluor 488 or 568-conjugated secondary antibodies(Invitrogen). Incubation with secondary antibodies alone did not giveany detectable background signal.

For capillary staining, tissue sections were incubated for 2 hours withFITC-conjugated Isolectin B4 (Lab Frontier) and Alexa-S68-Phalloidin(Invitrogen); a total of 26700 nuclei were analyzed overall on multiplesections of the border zone, of which 3300 for the assessment of CDCcontribution. Co-incubation of isolectin B4 with 500 mM galactose wasused as a negative control.

TUNEL staining was performed according to the manufacturer'sinstructions (In situ Cell Death Detection Kit, TMR red, Roche) andquantified on a total of 20500 nuclei in the border zone.

Confocal fluorescence imaging was performed on an Eclipse TE2000-Uequipped with a krypton/argon laser using UltraVIEW software (PerkinElmer). For image analysis of capillary and TUNEL slides, the ImageJsoftware was used for binary threshold of fluorescent images for eachchannel and consequent particle count. Masson's Trichrome staining wasperformed by standard methods. Briefly, staining was performed accordingto the kit manufacturer instructions (Sigma). High resolution imageswere acquired and processed with ImageJ software: color channels weresplit and the infarct area was manually traced on the blue channel.Threshold adjustment and area measurement functions allowed automaticcalculation of the collagen-stained fraction within the defined infarctregion.

Protein Array Screening of Conditioned Media

Analysis of serum free conditioned media by protein array analysisyielded 79 spots corresponding to various cytokines and growth factors.FIG. 18 shows two representative blots (18A) from cardiospheres and CDCsderived from the same patient sample, together with the correspondingdensitogram (18B and 18C), showing the cardiosphere/CDC optical densityratios for each factor. Three candidates were selected for furtheranalysis, VEGF, HGF and IGFI. This was based on their identity as nonimmune-modulatory factors, the high CSp/CDC ratio (suggesting enhancedsecretion in three-dimensional culture) and established roles in cardiacpathophysiology, particularly in myocardial infarction and heartfailure.

Regenerative Cells Release Growth Factors In Vitro

The various types of regenerative cells were analyzed to compare theirrelative paracrine potencies. Quantification of VEGF, HGF and IGF-1protein levels (by standard ELISA techniques) in the conditioned mediafrom cardiospheres, CDCs, and IICSps indicate that all threeregenerative cell types secrete significant amounts of these growthfactors. See FIG. 19A. During 48 hours of conditioning in low serum,cardiospheres released VEGF, HGF and IGFI. By contrast, CDCs and IICSpsreleased only VEGF in measurable amounts, although secretion by IICSpswas at the level measured in cardiospheres. In several embodiments,VEGF, HGF, and IGFI are all released, while in some embodiments, one ormore are released. In some embodiments, release of growth factors istime-dependent (e.g., one or more are released at an early time-point,while one or more are released at a later time-point).

Although different culture serum concentrations and different celldensities were also analyzed, IGFI secretion was not detected in CDCs orIICSps. CDCs are capable of releasing HGF, under certain cultureconditions. CDCs cultured in basal media release detectable amounts ofHGF as detected by ELISA (see FIG. 19B).

Immunofluorescent analysis of cardiospheres revealed ample VEGF, HGF andIGFI (FIG. 19C-19D). Reverse transcription PCR on RNA isolated fromcardiospheres, CDCs and IICSps also confirmed the expression of VEGF,HGF and IGFI mRNAs (FIG. 19E). No growth factors were detected by ELISAin normal human dermal fibroblast (NHDF)-CM in any of the conditionstested, although the corresponding mRNA was detected by PCR. NHDFs werealso grown as spheres on poly-D-lysine, but cell-associated growthfactors were not detected by immunofluorescence (data not shown) norwere secreted growth factors detected in CM by ELISA (FIG. 19A).

Cardiospheres and CDCs also express the receptors for VEGF, HGF and IGFI(respectively KDR, Met, IGFI-R), as assessed by immunofluorescence andRT-PCR (see FIG. 20).

Effects of Regenerative Cells Conditioned Media on Cardiac CellViability

Conditioned media from CDCs and NHDF cells were collected in fetalbovine serum-free basal media and used in place of NRVM media. After 24or 72 hours in 2% hypoxia, the percentage of early apoptotic NRVMs wasassessed by Annexin V/7-AAD labeling. No differences were detected after24 hours, however after 72 hours the percentage of early apoptotic NRVMswas dramatically lower in the CDC-CM (FIG. 21A) compared to the NHDF-CM(FIG. 21B) and the control FBS-free BM (FIG. 21C). The summary FACS datais represented in FIG. 21D. When CDC-CM was pre-incubated withneutralizing anti-VEGF and HGF antibodies and used for culturing NRVMsfor 72 hours, the apoptosis-reducing effect was significantly reduced(FIG. 21E). The neutralization of both secreted growth factors resultedin an excess 23% early apoptotic NRVMs compared to plain CDC-CM.Significance, in all examples, was evaluated using standard statisticaltechniques and significant differences are represented by p values<0.05, unless otherwise specified.

Effects of Regenerative Cells Conditioned Media on Angiogenesis

To determine if paracrine signals from the CDCs may be mechanisticallyinvolved in the blood vessel formation, HUVECs were cultured in eitherendothelial cell media (ECM; the normal media used to culture HUVECs),fetal bovine serum-free basal media, or CDC-CM (see FIGS. 21F, 21G, and21H, respectively). The ability of HUVECs to form complex tube networkswas lost in BM, but when cultured in CDC-CM this ability was almostcompletely recovered (compare 21G with 21H). The tube-forming ability ofHUVECs cultured in CDC-CM was significantly greater than that of HUVECscultured in NHDF-CM (FIG. 21J). Pre-incubation with anti-VEGF or bothanti-VEGF and anti-HGF neutralizing antibodies caused a slight butsignificant reduction of the total tube length per well (FIG. 21K).

In summary, the preliminary screening of human regenerative cells andtheir conditioned media revealed that these cell populations are capableof releasing many different cytokines and growth factors. Cardiosphereswere found to spontaneously release significant and higher amounts ofVEGF, HGF and IGFI in vitro as compared to CDCs. CDCs secrete only VEGFand HGF, although they maintain the transcription of all their mRNAs.However, IICSps were able to secrete VEGF at levels comparable tocardiospheres, suggesting the 3D structure of cardiospheres mayinfluence release of VEGF. This may be due to hypoxic stimulation of thecells residing in the interior layers of the cardiospheres.

These results suggest that: a) the primary CSp is the stage at whichparacrine abilities are maximal in vitro; b) VEGF release is affected bythe 3D structure; c) HGF and IGFI release fades with progressive time inculture. Furthermore, the expression of receptors for these growthfactors on regenerative cells suggests a possible autocrine feedbackeffect.

Although these results suggest that cardiospheres are more potent interms of paracrine signal release in vitro, other cell types may beequally, or more potent in a the more complex physiological environmentin vivo. To this end, functional in vitro analysis was performed to testthe effects of CDC-CM on cell viability and angiogenesis. The presentexperiments demonstrated that CDC-CM reduces the frequency of apoptosisinitiation in ventricular myocytes. This pro-survival effect ispartially due to synergy between secreted VEGF and HGF, as NRVMviability was reduced after pre-incubation of the CDC-CM with bothanti-VEGF and HGF neutralizing antibodies. CDC-CM has also potentpro-angiogenic effects, as demonstrated by promoting formation ofcomplex tube networks by HUVECs.

These data indicate that regenerative cells secrete growth factors thatpositively impact the survival of ventricular myocyte cells and theangiogenic capacity of endothelial cells. Thus, in several embodiments,these secreted growth factors play a role in the in vivo repair ofdamaged myocardial tissue by enhancing the survival of endogenous cellsand/or increasing angiogenesis in the myocardial tissue, among otherpossible mechanisms.

Detection of Human Growth Factors in Mouse Model

Western blot analysis using human-specific antibodies was used todetected engraftment of CDCs that were injected into murine cardiactissue as described above. Additionally, western blot was used todemonstrate the presence of human growth factors within the infarctedmouse heart. In several embodiments, growth factors from theadministered CDCs, such as VEGF, HGF, IGF-1 contribute to the functionalbenefits observed. Animals were sacrificed 1 day, 1 week or 3 weeksafter cell delivery. Regional tissue samples, ranging from 15 to 20 mgon average, were taken from infarct (INF), border zone (BZ), rightventricle (RV) and septum (SEP) areas for WB. Tissue samples were lysedin lysis buffer with proteinase inhibitors cocktail, homogenized, andspun. Supernatants were then collected and stored, after quantificationby Lowry assay of the protein content. Lysates were loaded on 4-12%Bis-Tris gels (Invitrogen), and blots 50 μg of protein per lane wasperformed with the Nupage mini-gels system (Invitrogen). Primaryantibodies against human VEGF and pan-GAPDH (Abcam), human HGF and humanIGF1 (R&D Systems), and human GAPDH (LabFrontier) were used. Membraneswere washed and incubated with HRP-conjugated secondary antibodies.Human growth factors were detectable with human specific antibodies onlyin lysates from CDC-injected animals, although hGAPDH was evident in theINF and BZ of both CDC- and NHDF-injected animals. These data indicatethe cells survive for at least three weeks in CDC-injected and for atleast 1 week in NHDF-injected hearts (see FIG. 22) Growth factors werealso detectable in remote areas, like the RV and SEP, 1 day after celldelivery. After 3 weeks, bands for hHGF and hIGF-1 were faint, butdetectable, although hVEGF could no longer be found detected. Theabsence of a hGAPDH band in the BZ after 24 hours can be attributed, atleast in part, to the difficulty in distinguishing the infarct from theperi-infarct zone at such an early time point.

Despite the apparent reduction in expression of human growth factorsover time, CDCs had clearly engrafted and survived, as evidenced by thedetection of hGAPDH in tissue lysates. These data suggest that, inseveral embodiments, CDCs exert an effect on the recipient tissue earlyafter transplantation, which has long lasting effects. In someembodiments, the engraftment of the CDCs itself triggers this effect.However, in several embodiments, paracrine effects are responsible forinitiating a cascade of events that lead to functional improvements atlater time point. In some embodiments, the initially administered CDCsare no longer present, yet the paracrine effects and the ensuing cascadeare still active in repairing and/or regenerating cardiac tissue. Inseveral embodiments, CDCs benefit the injured heart by direct myocardialregeneration with a large portion of their effect being due to thesecretion of paracrine factors that stimulate endogenous repairpathways. Based on a quantitative analysis of the contribution ofengrafted human cells (discussed in more detail below), it can beestimated that the paracrine effect accounts for at least 50% of thetotal effect, in one embodiment. In several embodiments, the paracrineeffect is responsible for about 50% to about 60% of the total effect,for about 60% to about 70% of the total effect, for about 70% to about80% of the total effect, or greater that 80% of the total effect. Insome embodiments, the paracrine effect is entirely responsible for themyocardial regeneration and/or increased cardiac function.

CDC-Injected Tissue Displays Higher Tissue Viability and CapillaryDensity

As discussed above, one week after cell delivery, human growth factorlevels remained high in the heart of mice injected with CDCs (see FIG.22). Lysates from CDC-injected mice contained higher levels of Aktprotein compared to NHDF-injected animals, as shown by WB and relativedensitometric analysis (FIG. 23A). Moreover, active Csp3 expression wasreduced in CDC-injected hearts relative to controls (FIG. 23B). Theseresults correlate with a reduced apoptotic rate (FIG. 23C) and highercapillary density in the border zone of CDC-injected mice (FIG. 23D),compared to controls, as assessed by TUNEL and isolectin B4 staining,respectively. Taken together, these data indicate that CDCs suppresspost-ischemic apoptosis and improve blood supply. Thus, in severalembodiments, reduction in apoptosis accounts for at least a portion ofthe higher tissue viability. In several embodiments, increasedangiogenesis, alone or in combination, with reduced apoptosis, accountsfor the increased tissue viability.

Direct Versus Indirect Regenerative Contribution

As a means of quantifying how much of the functional improvement due toCDC administration is related to direct regeneration versus indirectparacrine effects, the relative contribution of human CDCs to thecapillary density in the tissue areas where CDCs were detectable after 1week was calculated. Despite an overall doubling of capillary density inCDC-injected mice, only 9.6±2.7% of the total capillaries were found tobe of human origin (FIGS. 24A and 24B). This amounts to ˜20% of theenhanced angiogenesis; thus, the angiogenic effect reflects both directregeneration and paracrine effects, with latter predominating.

With respect to the cardiomyogenic effect of CDC transplantation, after1 week the infarct area of CDC-injected mice contained a higherpercentage of viable myocardium (FIG. 25A), as assessed by Masson'strichrome staining Within those viable areas, 11.8±4.5% of theMHC-expressing cells were of human origin (FIGS. 24C and 24D). Whilesignificant, this explains only half of the ˜20% increase in relativetissue viability revealed by Masson's trichrome staining, and only ˜25%of the overall doubling in MHC nuclei (FIG. 24C). Thus, both directregeneration and paracrine effects underlie the cardiomyogenic effectsof CDC transplantation. No human capillaries or MHC-positive cells weredetectable in the NHDF-injected hearts.

This Example has therefore demonstrated that when injectedintramyocardially in infarcted SCID mice, CDCs release, among othergrowth factors, VEGF, HGF and IGFI. These growth factors are detectablefor at least 1 week after cell. Growth factor release is not simply aresult of cell administration or engraftment, as both CDCs and NHDFcells engrafted into cardiac tissue, rather growth factors secreted bytransplanted cells were detectable only in CDC-injected animals.Additionally, only CDC-injected animals demonstrated a significantfunctional improvement.

Growth factors were detected in remote areas of the heart 24 hours aftercell injection. Remote areas did not appear to contain growth factorsafter 1 week, suggesting that diffusion of growth factors occurs at theearlier time point. This may be due to the relatively intact vascularsystem at the earlier time, which loses functionality in the more matureinfarct scar 1 week after surgery, thus restraining human growth factorsto tissue more local to the CDC engraftment zone. Alternatively, theearly spread into neighboring areas may simply reflect a greaterintensity of growth factor production soon after injection. This may bedue to a higher production rate by newly-injected cells, or may be dueto the presence of more cells at early post-injection time points.

Cell survival, particularly in an unfavorable environment (such asischemic cardiac tissue), mostly depends on the cells' ability toovercome death triggers and secondarily to promote angiogenesis. In thisrespect, the in vitro anti-apoptotic and pro-angiogenic effectsdemonstrated in the prior example correlate with the in vivo observationthat, 1 week after cell delivery, Akt was up-regulated in tissue samplesfrom CDC-injected animals. Contemporaneously, the active form of theapoptotic effector Csp3 was less expressed in CDC— compared toNHDF-injected hearts (FIG. 23B). Furthermore, the rate of TUNEL-positivecells was significantly reduced in the border zone of CDC-injected mice,which also displayed higher capillary density compared to controls (FIG.23C-23D). Overall, these results imply that the injection of CDCs ininfarcted SCID mouse hearts favors higher tissue viability in theinfarct and peri-infarct areas, and consistently correlates with reducedapoptosis and improved blood supply. This conclusion is consistent withprevious histological data showing a higher percentage of viablemyocardium in the infarct area of CDC-injected, as compared toNHDF-injected mice (which was also confirmed at the 1 week time point inthe present study).

While injected CDCs and/or their progeny persist for at least threeweeks post-administration, in several embodiments the persistence ofCDCs reflects their multilineage differentiation. In severalembodiments, the persistence of CDCs reflects a survival advantageconferred by paracrine effects such as those described here. In severalembodiments a combination of both mechanisms is at play. The presentstudy demonstrates that CDCs directly contribute to approximately 10% ofthe overall capillary density in the areas of CDC engraftment. However,there remains a statistically significant difference between thecapillary density of the CDC and the NHDF groups, confirming the majorrole of indirect paracrine induction by CDCs.

With respect to their contribution to cardiomyogenesis, directregeneration and paracrine mechanisms supporting regeneration appearedto be more equivalent in their effects. Given that long-term engraftmentof transplanted cells is low, it seems reasonable to focus on enhancedengraftment as a prime strategy to boost the overall efficacy of celltherapy, even if the final benefit reflects both direct and indirectregeneration.

While the use of cytokines and growth factors as cardiac therapeutictools has been carefully investigated in the past few years, especiallyby means of gene therapy, many such protocols involve risks such aspathologic angiogenesis, severe inflammatory reactions and arrhythmias.The possibility of growth factor release under biological andphysiological control through regenerative cell therapy might offer anideal combination of strategies in that regenerative cells are able todifferentiate directly and to concurrently secrete beneficial moleculesand harness endogenous repair.

Direct injection of HGF and IGF-1 into the myocardium has been shown tosuccessfully mobilize endogenous cardiac stem cells. Given that CDCtransplantation produces these same growth factors, as shown herein,this study suggests that recruitment of endogenous stem cells is alikely contributor to the functional improvement demonstrated in thisstudy.

These data, taken together, demonstrate that regenerative cells secretesignificant amounts of pro-survival and pro-angiogenic growth factors invitro, and in an in vivo cardiac cell therapy model. This Role ModelEffect appears to be a key mechanism, together with the naturalpropensity of regenerative cells for cardiac differentiation,contributing to the regenerative potential and therapeutic effects ofregenerative cells in the post-infarction period.

Efficacy of CDC Cell Banks in Mouse Model

Based on the discussion above regarding the generation of cell banks,which are used in some embodiments, the following sets forth an exampleof a contemplated experiment using allogeneic cells generated and bankedprior to use. The same mouse MI model will be employed. CDCs having gonethrough the manufacturing process described above, includingintermediate banking, will be suspended and delivered in acryopreservation solution (CRYOSTOR® CS5). Also as discussed above,CRYOSTOR® CS5 used, in some embodiments as an excipient that is combinedwith the CDCs. Control groups will consist of mice injected withCRYOSTOR® CS5 alone, mice injected with CDCs suspended in PBS, and miceinjected with PBS alone. In addition to measuring function 3 weekspost-MI, histology will be performed to assess the degree of localtissue damage that may occur due to the 5% dimethylsulfoxide (DMSO)contained in the cryopreservation solution.

Example 11 Immunologic Properties of Regenerative Cells

In order to understand the potential for cardiac tissue repair viaallogeneic transplant of regenerative cells, the immune responsesinitiated against allogeneic regenerative cells was characterized. Bothin vitro and in vivo analyses were performed using an established ratmodel. CDCs were isolated from two rat strains with different MHChaplotypes (Wistar Kyoto and Brown Norway). Cross-transplantation oforgans from these mismatched strains has been used as a model forallograft rejection. CDCs were isolated from Wistar Kyoto rats and exvivo expanded, as described above. These CDCs were intramyocardiallyinjected into either Wistar Kyoto rats (syngeneic/autologous model) orBrown Norway rats (allogeneic model). In some rats, human CDCs wereinjected (xenogeneic model). Expanded CDCs (or PBS for control groups)were injected immediately after MI was simulated via LAD ligation, asdescribed above. Injections comprised 2×10⁶ CDCs in 100 μL totalinjection volume (50 μL at each of two peri-infarct sites). In alltransplants, male Wistar-Kyoto rats were the donors and female rats werethe recipients, thus enabling mRNA isolated from rats post-injection tobe used in quantitative y-chromosome real time PCR reactions todetermine engraftment of cells into the target tissue.

Rats underwent echocardiography to assess efficacy, were subjected toblood draws to assess the level of pro- and anti-inflammatory cytokinespresent in the serum, and were euthanized at the end of the study forhistology to examine the degree of immune rejection evident or PCR toquantify the percentage of CDCs engrafted.

In Vitro Immunologic Properties of Allogeneic Cdcs

The immunologic properties of rat and human CDCs (rCDCs and hCDCs,respectively) were first examined in vitro. Established flow cytometrymethods were used to assess the expression of MHC Class I and Class IIantigens and before and after stimulation with interferon-γ, a knownimmunostimulatory molecule, for 1 day or for 5 days. With regard toimmune antigens, both rCDCs and hCDCs express MHC I but not MHC class IIsurface antigens (FIGS. 26B and 26F). Incubation with interferon-γupregulated both MHC I and MHC II expression in a time-dependent manner(FIG. 26C/D and 26G/H). CD80/CD86 are expressed on antigen presentingcells and provide co-stimulatory signals needed for T cell activationand survival. Expression of both CD80 and CD86 was relatively low inboth rCDCs and hCDCs at baseline. Stimulation with interferon-γ did notsignificantly induce expression of these co-stimulatory molecules (seee.g., FIG. 26I). Several other markers did not differ in expressionlevels between rCDCs and hCDCs (see e.g., FIG. 26J).

The observed baseline immunophenotype of CDCs is beneficial forallogeneic applications. In several embodiments, the higher baselinelevels of MHC class I antigen expression are beneficial, as thisantigen, at least in part, protects the cells from natural-killercell-mediated destruction. Similarly, the low level expression of MHCclass II antigens (potent activators of the immune system) allows, inseveral embodiments, allogeneic CDCs to escape direct recognition fromCD4+ T helper cells. While MHC class I antigens may activate effector Tcells in certain cases, the low quantities of costimulatory molecules(CD80/CD86) would induce an interruption in the signaling pathway,thereby likely leaving T cells inactive.

Lymphocyte proliferation was measured by BrdU incorporation. Well-knownmethods were used to evaluate immunogenicity, which are described inbrief. Spleens were harvested aseptically from euthanized WKY and BNrats, mechanically dissociated and filtered through a 100 μm nylon mesh.Erythrocytes were lysed with 0.83% ammonium chloride, cells were washedin RPMI 1640, dead cells were removed by density centrifugation and cellviability was assessed by trypan blue dye exclusion. Stimulating rCDCsand hCDCs were mitotically inactivated with 50 μg/ml mitomycin C(Sigma-Aldrich) in the dark at 37° C. for 30 minutes and washed threetimes with RPMI 1640. 10⁴ stimulating CDCs were cocultured with 10⁵responderlymphocytes in 200 μl of culture medium (RPMI 1640 supplementedwith 10% FBS) in 96-well flat-bottom plates for 5 days. The followingexperimental conditions were tested in quadruplicates: a) rCDCscocultured with WKY lymphocytes (syngeneic coculture); b) rCDCscocultured with BN lymphocytes (allogeneic coculture); c) hCDCscocultured with BN lymphocytes (xenogeneic coculture). All appropriatecontrols were also tested. BrdU was added to the cocultures for the last24 hours and responder cell proliferation was assessed by the CellProliferation Biotrak ELISA System (GE Healthcare) according to themanufacturer's instructions. Absorbance was measured with a microplatereader (Bio-Rad) at 450 nm. Alloreactive and xenoreactive lymphocyteproliferation is presented as relative proliferative response,normalized to syngeneic coculture proliferation (stimulation index). Thecell-free supernatant of the cocultures was collected and the levels ofsecreted IFN-g, IL-1b, IL-13, IL-4, IL-5, KC/GRO and TNF-α were measuredby electrochemiluminescence. The levels of secreted IL-2 were measuredusing enzyme-linked immunosorbent assay (ELISA) kits, according to themanufacturer's protocols (R&D Systems).

Co-culture of rat CDCs with allogeneic lymphocytes induced negligiblelymphocyte proliferation, which was comparable to that induced bysyngeneic CDCs and less than that of xenogeneic human CDCs (FIG.26K-26M). The lymphocyte proliferation induced by allogeneic CDCs wasnot significantly different than that induced by syngeneic CDCs(syngeneic stimulation index: 1.4±0.2; p=ns versus allogeneic). Incontrast, xenogeneic hCDCs induce a strong proliferative response(stimulation index: 2.6±0.6; p<0.01 vs. syngeneic or allogeneiccocultures; FIG. 26N). Quantification of inflammatory cytokines in thecoculture supernatants by electrochemiluminescence and ELISAdemonstrated comparable levels of pro-inflammatory (IFN-γ, TNF-α, IL1b,IL2, KC/GRO) and anti-inflammatory (IL5, IL13, IL4) cytokines insyngeneic and allogeneic cocultures. Again in contrast, in thexenogeneic cultures, secretion of all inflammatory cytokines wasmarkedly increased, indicating significant activation of responderlymphocytes (FIG. 26O). Consistent with these in vitro results, the invivo immune response (discussed in more detail below), was notsignificantly different between allogeneic and syngeneic celltransplants.

Engraftment of Allogeneic CDCs in Rat Model

To enable measurements of cell engraftment in vivo, CDCs were transducedwith a lentiviral construct expressing GFP. Quantitative PCR wasperformed 1 week and 3 weeks post cell injection in order to monitortransplanted cell engraftment after syngeneic, allogeneic and xenogeneiccell transplantation. Cells isolated from male donor WKY rats and malehuman biopsies were injected into the myocardium of female recipientsand quantified absolute cell engraftment by real-time PCR using the (ratand human respectively) SRY gene located on the Y chromosome as target.In brief, the recipient heart was explanted, weighted, homogenized andgenomic DNA was isolated using the DNA Easy minikit (Qiagen), accordingto the manufacturer's protocol. The TaqMan® assay (Applied Biosystems)was used to quantify the number of transplanted cells with the rat (forsyngeneic and allogeneic transplantation) and human (for xenogeneictransplantation) SRY gene as template. A standard curve was constructedwith samples derived from multiple log dilutions of genomic DNA,isolated from male rat hearts and samples of male human myocardium,spiked with 50 ng of female rat genomic DNA as control. The copy numberof the SRY gene at each point of the standard curve was calculated basedon the amount of DNA in each sample and the total mass of the rat genomeper diploid cell. All samples were tested in triplicates. For eachreaction, 50 ng of template DNA was used. The result from each reaction,copies of the SRY gene in 50 ng of genomic DNA, was expressed as thenumber of engrafted cells/heart by extrapolation to the total DNAcontent of each heart, taking into account that there is one copy of theSRY gene per transplanted cell. FIG. 27A depicts the experimental schemeemployed to study engraftment and function. FIG. 27B describes theexperimental and control groups used to evaluate engraftment andfunction.

Two million male syngeneic, allogeneic or xenogeneic CDCs were implantedinto the ischemic myocardium of female rats, immediately after LADligation. Similar to the immunogenicity results discussed above (e.g.,the limited differences in immune induction between syngeneic andallogeneic cultures) engraftment of allogeneic and syngeneic CDCs wasnot significantly different between allogeneic and syngeneic transplantsat 1 week post MI (absolute cell number: 117,587±94181 vs.122,662±68,637; p=ns). In contrast, the majority of xenogeneic CDCs failto engraft within 1 week of transplantation (absolute cell number:10,535±4012; p<0.05 compared to syn, allo groups). Data related to1-week engraftment are shown in FIG. 27C.

Three weeks after experimental infarction and CDC delivery, cellengraftment decreased markedly (to <1% of cells transplanted) in bothsyngeneic and allogeneic groups. However, the residual number ofengrafted cells is higher after syngeneic transplantation (absolute cellnumber: 13,343±6427 vs. 3169±4012; p<0.05). This likely reflects gradualdestruction and clearance of the allogeneic cells by the host immunesystem. No engrafted xenogeneic cells were detected after 3 weeks. Dataare presented in FIG. 27D. These results indicate, that, in someembodiments, allogeneic CDCs are cleared more rapidly than syngeneicCDCs between days 8 and 21 post-delivery. In some such embodiments, thebeneficial effects of allogeneic CDCs have already been realized (or putin motion), such that the survival and residency of the delivered cellsis no longer necessary. Moreover, based on the immunogenicity datadiscussed above, these data indicate that the immune response is notsolely responsible for the decreased number of engrafted cells.Endogenous mechanisms to remove transplanted cells, and/or the naturallifespan of the administered cells may account for the decrease.

Immune Response to Allogeneic CDCs in Rat Model

Consistent with the limited in vitro induction of pro-inflammatorycytokines by allogeneic CDCs, administration of allogeneic CDCs inducedonly a mild local immune reaction in the heart. In fact, the immunereaction is barely visible using a standard H&E stain (example figuresshown at 3 weeks post-MI, FIG. 28B-28C). Significant infiltration isseen in the xenogeneic transplant (FIG. 28A). Further, using anestablished pathological assessment scale, the ISHLT grading system,which is used in clinical practice to diagnose rejection, the level ofrejection seen in H&E sections were scored at 1 week, 3 weeks, and 6months post-MI. Four to five animals and 48-60 sections were scored ineach group (Grade 0=No rejection, Grade 1R=Interstitial and/orperivascular infiltrate with up to one focus of myocyte damage, Grade2R=Two or more foci of infiltrate with associated myocyte damage, Grade3R=Diffuse infiltrate with multifocal myocyte damage). The analysis wasconducted by a blinded cardiac pathologist. No significant immunerejection could be detected in the allogeneic setting at any time point(see 28B and 28D, data for 6 weeks not shown). In contrast, xenogeneiccell transplantation resulted in significant mononuclear infiltrationthat could be detected in the infarct scar and border zone 1 week (FIG.28D) and 3 weeks (FIG. 28F) post treatment. As shown in FIG. 28A, theinfiltrating cells were located at interstitial and perivascular spaces,but notably, no foci of myocyte damage could be detected. The lack offoci suggests that immune rejection of xenogeneic CDCs did not inflictadditional damage to the myocardium. The immune response was resolved inall groups at 6 months post-MI.

Despite its utility in the clinical assessment of transplant rejection,detection of small foci of rejection by H&E staining is complicated in apost-MI setting due to the natural inflammatory response to the ischemicinsult. In order to corroborate the histochemical data, immunostainingagainst a variety of immune cell markers was performed for each timepoint. This approach allows a characterization/identification of theinfiltrating inflammatory cells. After allogeneic transplants,immunohistochemistry revealed rare events of rejection. A few small andsparse infiltrates around some transplanted cells were detected 3 weekspost-treatment. See for example FIG. 28H, 28I, and 28J. Theseinfiltrates comprised mainly CD3+ T lymphocytes (with equalcontributions of CD8+ T cytotoxic and CD4+ T helper subpopulations) andto a lesser extent CD45R+B lymphocytes and CD11c+ dendritic cells (seeFIG. 28K ₁-28K₁₅ and also 29A-29C, which depict syngeneic, allogeneic,and xenogeneic transplants, respectively). Based on the similarquantities of CD4+ and CD8+ T lymphocytes in the graft area, as well asthe presence of dendritic cells, it is possible that an indirect pathwayof allorecognition plays a greater role in the immune rejection oftransplanted cells. For example, antigens shed by apoptotic donor CDCsmay be phagocytosed by host antigen presenting cells (like dendriticcells) and subsequently presented to CD4+ cells, thus activating theimmune cascade. However, in some cases, participation of the directpathway of allorecognition also likely plays a role. Notably, theincreased lymphohistiocytic infiltration was significantly lower thanthat seen with xenogeneic transplantation (FIGS. 28L and 28M; and alsoFIGS. 29D and 29E), and had completely resided by 6 months (data notshown). The higher infiltration of CD68+ macrophages (which in generaldid not localize within the infiltrates but were evenly dispersed alongthe infarct) detected at 1 and 3 weeks post MI in the xenogeneic andcontrol groups was consistent with the larger infarct size observed inthose groups.

Furthermore, there were no significant signs of systemic immunogenicity(FIGS. 30A-30G) in the animals who received allogeneic CDCs as evidencedby serum concentrations of pro-inflammatory cytokines IFN-γ, IL-10,KC/GRO, TNF-α, (FIGS. 30A, 30B, 30F, and 30G, respectively), or theanti-inflammatory cytokines IL-13, IL-4, and IL-5 (FIGS. 30C, 30D, and30E, respectively). In contrast, in the xenogeneic transplants, thecirculating levels of IFN-γ, IL1β, IL13 and IL4 were markedly increased.Taken together, these data indicate that the systemic inflammatoryresponse observed after xenogeneic transplantation did not occur in theallogeneic setting. Thus, in some embodiments, allogeneic transplantsprovide the unexpectedly beneficial combination of functional andmorphological improvements in damaged cardiac tissue, without theexpected immune response. For these reasons, among others disclosedherein, allogeneic cells are particularly advantageous in someembodiments.

Allogeneic CDCs Elicit a Cellular but not a Humoral Response Memory

In order to assess the development of cellular memory immune responseafter allogeneic CDC transplantation, the alloreactivity of lymphocytesisolated from spleens of allogeneic recipients 3 weekspost-transplantation was assessed by one-way mixed lymphocyte reactions.Lymphocytes from sensitized animals exhibited higher proliferation aftercoculture with allogeneic CDCs (stimulator index 2.32±0.52), compared tonaïve lymphocytes (p<0.05) or syngeneic cocultures (p<0.01). Inaddition, markedly increased levels of inflammatory cytokines in thesupernatants of sensitized lymphocyte cocultures were detected byelectrochemiluminescence and ELISA. These results are indicative of a Tcell memory response and are in accordance with the immunohistochemistrydata discussed below, which shows a predominant role of T cells in thesparse mononuclear infiltrates observed 3 weeks post allogeneictransplantation (FIG. 28C).

In order to assess the development of a humoral memory response,recipient rat sera obtained 1 and 3 weeks post-transplantation werescreened for circulating anti-donor antibodies. No alloreactiveantibodies were detected in any recipients of allogeneic CDCs at anytimepoint. This finding is in contrast to the xenogeneic transplants inwhich high titers of xenoreactive IgM antibodies were detected 1 and 3weeks post transplantation. Additionally, a progressive increase ofxenoreactive IgG antibodies was observed from week 1 to week 3. Thedevelopment of anti-donor antibodies in xenogeneic recipients, but notin allogeneic recipients, is consistent with the significantly higher(−8 fold) B cell myocardial infiltration observed in the xenogeneicsetting (FIGS. 28L-28M).

Efficacy of Allogeneic CDCs in Rat Model

Morphometric analysis of explanted hearts 3 weeks post infarction showedsevere LV chamber dilatation and infarct wall thinning in animals in thexenogeneic and control groups (FIG. 31A, bottom row). In contrast, thesyngeneic and allogeneic groups exhibited smaller scar size, increasedinfarcted wall thickness and attenuation of LV remodeling (FIG. 31A).Scar size and infarcted wall thickness did not differ among animalstreated with syngeneic or allogeneic CDCs, which indicates, in someembodiments, that similar physical (e.g., treatment effects are obtainedwhether autologous or allogeneic cells are used. (FIGS. 31B-31C).

To assess functional benefit of CDC transplantation, global cardiacfunction was assessed by echocardiography, quantifying fractional areachange (FAC), left ventricular ejection fraction (LVEF), and fractionalshortening (FS). At baseline (DO), FAC, LVEF and FS did not differ amongtreatment groups, indicating a similar degree of initial injury. Overthe first 3 weeks after infarction, indices of function failed toimprove in the xenogeneic and control groups, whereas FAC, LVEF and FSall rose significantly in both the allogeneic and syngeneic groups(FIGS. 31D-31F). Notably, the functional benefit observed at 3 weekspersisted out to 6 months post infarction in the allogeneic andautologous groups, but not in xenogeneic or control group. A treatmenteffect summary is shown in FIG. 31G, which show that, despite theirlower engraftment at 3 weeks, allogeneic cells are equivalent tosyngeneic (which are modeling autologous cells in this experiment) interms of providing functional repair to damaged cardiac tissue

Both syngeneic and allogeneic CDCs led to similar improvements incardiac function in the rat model 3 weeks, 12 weeks, and 6 monthspost-MI (FIG. 31). Both syngeneic and allogeneic groups differedsignificantly from control animals at each timepoint. The xenogeneicgroup showed a decline in function at 3 weeks post-MI that issignificantly different from the result seen in the syngeneic andallogeneic groups. This study indicates that allogeneic CDCtransplantation without immunosuppression is safe and improves heartfunction in a rat model without the need for persistent cellengraftment.

To further investigate possible mechanisms of the benefits provided byallogeneic transplants, the fate of the transplanted cells themselves aswell as indirect mechanisms of benefit were evaluated.Immunohistochemistry revealed that syngeneic and allogeneic CDCsprimarily resided in the border zone and infarct scar. A subset ofadministered cells were found have reentered the cell cycle at 1 and 3weeks post-MI, as indicated by Ki-67 positivity and BrdU incorporation.Rare events of cardiomyogenic (GFP+/αSA+ cells) and angiogenic(GFP+/vWf+ cells) differentiation of surviving CDCs could be detected inboth the syngeneic and the allogeneic setting. While the majority ofGFP+/αSA+ cells were small and exhibited an immature cardiomyocytephenotype (FIG. 31H), mature GFP/αSA+ cells structurally integrated intothe host myocardium could also be detected (FIG. 31I, white arrow). Inaddition, GFP+/vWf+ cells were found to be incorporated in microvesselsin the risk region (FIG. 31J, white arrows). These results demonstratethe multilineage potential of CDCs, e.g., that CDCs can generate thevarious cell types needed for complete cardiac repair. However, directdifferentiation of administered cells was low, and thus, in someembodiments, unlikely to fully account for the observed robustfunctional benefit of allogeneic cell transplant. Thus, in someembodiments, the direct effect of administered cells is only partiallyresponsible for the observed benefits (e.g., physical and functionalcardiac repair).

Endogenous cardiac regeneration is another possible mechanism and wastherefore evaluated. Endogenous regeneration may involve one or more ofendogenous cardiomyocyte cell cycle re-entry, recruitment of endogenousprogenitor cells to the site of cell transplantation, and/or enhancedangiogenesis. Both syngeneic and allogeneic CDC therapy stimulatedresident cardiomyocyte cell-cycle re-entry. The number of cycling hostcardiomyocytes (GFP−/αSA+/Ki67+ and GFP−/αSA+/BrdU+ cells) was markedlyincreased in CDC-treated hearts compared to controls (FIGS. 31K-31M,31N-31P, 31W, and 31X) at 1 and 3 weeks post MI. However, the number ofGFP−/αSA+/Ki67+ and GFP−/αSA+/BrdU+ cells significantly decreased from 1week to 3 weeks, dropping to nearly undetectable levels at 6 months,suggesting that, in some embodiments, the re-entry of endogenous cardiaccells into the cell cycle is perhaps an acute effect. In otherembodiments, however, this effect may be longer-lasting. Syngeneic andallogeneic CDC transplantation also recruited endogenous stem cells(FIG. 31Q-31S and 31Y) as evidenced by the increased number ofGFP−/c-Kit⁺ in CDC-treated hearts compared to controls at 1 week and 3weeks post MI. As with resident cycling myocytes, the number ofendogenous progenitors significantly decreased as a function of time.

Finally, it was determined that both syngeneic and allogeneic CDCtransplantation enhanced angiogenesis in the infarct border zone. Vesseldensity, identified by immunostaining for vWf, was markedly increased 3weeks after cell therapy compared to controls (FIGS. 31T-31V and 31Z).While control hearts did display some activity in these endogenousreparative mechanisms, both syngeneic and allogeneic showed a greatermagnitude than that of control cells.

These data thus indicate that either syngeneic or allogeneic transplantsyield functional improvements, while control groups lose cardiacfunction over time. Additionally, in vivo data evaluating infarct sizeindicate that infarct size is significantly smaller in both cell therapygroups compared to controls. See, e.g., FIG. 25. Additionally, the datapresented herein suggests that exogenous CDC administration stimulatesactivation of endogenous repair and/or regeneration pathways. Thus,indirect mechanisms may be, in some embodiments, largely responsible forthe observed benefit following CDC therapy.

To further investigate the indirect effects, myocardial levels ofbeneficial paracrine factors in the infarct border zone were analyzed.Western Blot analysis revealed increased secretion of VEGF, IGF-1 andHGF in hearts treated with syngeneic and allogeneic CDCs, compared tocontrols, at day 1, day 4 and day 7 post MI (FIG. 31AA-DD). 3 weeks postMI, no difference in secretion of these factors could be observed amonggroups. This indicates that syngeneic and allogeneic CDCs are equivalentin terms of their generated paracrine effects, both in magnitude and intime course. In some embodiments, the factors evaluated above areprimary players in the indirect repair effect, however, in someembodiments, other factors (described elsewhere herein) may also play arole.

Both rat and human CDCs show similar patterns of MHC expression atbaseline and after IFN stimulation. As with the human CDCs, IFNstimulation induces upregulation of rat CDC MHC I, II molecules.Co-culture of rat CDCs with allogeneic lymphocytes induces lymphocyteproliferation and secretion of pro-inflammatory cytokines However, thelevel of immune response is significantly lower when compared toxenogeneic co-culture.

In vivo, rat syngeneic and allogeneic CDCs demonstrate similar survivalrates at Day 8 after experimentally induced MI. At Day 21, cell survivalafter syngeneic transplantation is significantly higher. This suggeststhat the pro-immunogenic characteristics that CDCs display in vitro,induce an in vivo immune response between day 9 and day 21. Regardless,overall cell survival is poor in both syngeneic and allogeneic groups.It is possible, that more than one mechanism of cell death is involved,depending on the group. For example, higher levels of apoptosis may beinvolved in reducing the overall survival of the allogeneic cells whilea lesser amount of apoptosis couples with increased necrosis may accountfor cell loss in the syngeneic group.

Despite the significantly reduced number of cells present after 21 days,both syngeneic and allogeneic CDC transplantation led to significantimprovement of LV function after MI, as compared to controls. Thetreatment effect is similar between the two types of transplants at day21. Moreover, infarct size is reduced in the two groups as compared tocontrols. This suggests that, as discussed above, an early, butlong-lasting effect is induced by the transplant of cells into damagedmyocardium. It is possible that the paracrine effects described in theprior Examples are responsible for inducing a cascade of events thatserve to improve cardiac function by repairing damaged tissue orgenerating new, functional tissue. It is also possible that these earlysignals recruit endogenous cardiac stem cells that augment cardiacfunction and induce tissue repair. In either case, the data presentedhere indicate that the presence of viable transplanted cells is not anecessary precursor to improved cardiac function and/or tissue repair.It appears that the transplanted cells function as a trigger, and thus,in some embodiments, induce and/or recruit repair mechanisms, then areremoved by various cell death pathways, including, but not limited tophagocytosis, autophagy, apoptosis, enzymatic degradation, among others.In this light, the allogeneic cell transplant becomes even moreattractive, not only because of the practical advantages discussedabove, but because the life span of the transplanted cells appears to beshort enough that the induced immune responses are not significantenough to interfere with functional repair of the damaged tissue.

Example 12 Pre-Clinical Trials Using Autologous Cells

Cardiosphere-derived cells (CDCs) and their 3-dimensional precursors,cardiospheres were tested, according to several embodiments of theinvention, for cellular cardiomyoplasty in a mini-pig model of heartfailure post-myocardial infarction (MI). Although porcine studies wereconducted, data can be extrapolated to human patients according toseveral embodiments of the invention.

According to one embodiment, autologous cardiospheres or CDCs grown fromendomyocardial biopsies were injected via thoracotomy four weekspost-anteroseptal MI. Engraftment optimization with luciferase-labeledCDCs guided the choice of cell dose (0.5M cells/site) and target tissue(20 periinfarct sites). Pigs were randomized to placebo (n=11),cardiospheres (n=8) or CDCs (n=10). Functional data were acquired beforeinjection and again 8 weeks later, after which organs were harvested forhistopathology. Beyond the immediate perioperative period, all animalssurvived to protocol completion. Ejection fraction was equivalent atbaseline but, at 8 weeks, was higher than placebo in both of thecell-treated groups (placebo vs. CDC p=0.01; placebo vs. cardiospheresp=0.01). Echocardiographic and hemodynamic indices of efficacy improveddisproportionately with cardiospheres. Likewise, adverse remodeling wasmore attenuated with cardiospheres than with CDCs. Provocativeelectrophysiologic testing showed no differences among groups, and notumors were found.

Thus, according to several embodiments, dosage-optimized directinjection of cardiospheres or CDCs is safe and effective in preservingventricular function in ischemic cardiomyopathy. In one embodiment, CDCsand cardiospheres have equivalent effects on LVEF. In some embodiments,cardiospheres are superior in improving hemodynamics and regionalfunction, and in attenuating ventricular remodeling.

In several embodiments of the invention, the regenerative cellsdelivered to patients are cardiospheres. In other embodiments, theregenerative cells are CDCs. In yet other embodiments, the regenerativecells are a combination of cardiospheres and CDCs. In some embodiments,regenerative cells are useful for treating dysfunction (e.g., leftventricular dysfunction) post-MI.

In some embodiments, CDCs delivered to subjects (e.g., non-humanmammals, human patients) are a heterogeneous mix of cells expanded fromcardiac tissue, with formation of self-assembling spherical clusters ofheart-derived cells (cardiospheres) as an intermediate processing step.In several embodiments, CDCs are clonogenic and exhibit multi-lineagepotential, thus fulfilling key criteria for cardiac stem cells, and theycan be readily and reliably expanded from tiny specimens of heartmuscle. According to one embodiment, approximately 20 mg samples yieldabout 1.5 million CDCs on average within 45 days.

In one embodiment, cardiospheres having a size of about 50-200 μm indiameter are used. In one embodiment, delivery mechanisms other thanintracoronary administration are used to reduce the risk of embolizationat the arteriolar level. For example, in one embodiment, cardiospheresare delivered by intramyocardial injection. In one embodiment,cardiospheres having a size of about 50-150 μm in diameter are used. Inseveral embodiments, CDCs are particularly advantageous due to theirsize being less than that of cardiospheres. In some embodiments, CDCsare of a size that is associated with little to no risk of embolizationof at the arteriolar level. For example, in several embodiments, CDCsare less than about 50 μm in diameter. In some embodiments, CDCs areless than about 40 μm in diameter, less than about 30 μm in diameter,less than about 20 μm in diameter, and less than about 10 μm indiameter. In some embodiments, CDCs range from about 5-10 μm indiameter, about 6-11 μm in diameter, about 7-12 μm in diameter, about8-13 μm in diameter, about 9-14 μm in diameter, about 10-15 μm indiameter, about 11-16 μm in diameter, about 12-17 μm in diameter, about13-18 μm in diameter, about 14-19 μm in diameter, about 15-20 μm indiameter, and overlapping ranges thereof. In some embodiments, CDCs areless than about 75% of the size of a cardiosphere. In some embodiments,CDCs are less than about 50% of the size of a cardiosphere. In someembodiments, CDCs are less than about 25% of the size of a cardiosphere.In some embodiments, CDCs range from about 75% to about 70% of the sizeof a cardiosphere, from about 70% to about 65% of the size of acardiosphere, from about 65% to about 60% of the size of a cardiosphere,from about 60% to about 55% of the size of a cardiosphere, from about55% to about 50% of the size of a cardiosphere, from about 50% to about45% of the size of a cardiosphere, from about 45% to about 40% of thesize of a cardiosphere, from about 40% to about 35% of the size of acardiosphere, from about 35% to about 30% of the size of a cardiosphere,from about 30% to about 25% of the size of a cardiosphere, from about25% to about 20% of the size of a cardiosphere, from about 20% to about15% of the size of a cardiosphere, from about 15% to about 10% of thesize of a cardiosphere, from about 10% to about 5% of the size of acardiosphere, from about 5% to about 1% of the size of a cardiosphere,and overlapping ranges thereof. In several embodiments, CDCs areparticularly advantageous because of their size, which, being less thanthat of cardiospheres, enables a greater degree of engraftment, which,in some embodiments, increases the beneficial effects of CDCadministration, whether due to the engraftment itself or an increasedparacrine effect related thereto. It shall be appreciated, that thesource of the CDCs, be it an autologous, xenogeneic, or allogeneicsource, does not substantially impact the advantages realized in certainembodiments due to the smaller size of CDCs.

Two studies were performed to examine regenerative cells according toseveral embodiments of the invention. Study 1 (FIG. 32A) consisted ofopen-label experiments to quantify engraftment 24 hours afterintramyocardial CDC injection in the porcine MI model. The engraftmentdata were used to inform the dosage and target tissue of injection ofCDCs for Study 2 which was a pivotal placebo-controlled, blindedrandomized study of safety and efficacy of either cardiosphere or CDCdirect intramyocardial injection. (FIG. 32B).

Animals were randomized to receive either placebo or about 10 millioncells in cardiosphere or CDC form, administered as 20 injections of 0.5million per site. Other cell and/or injection numbers are used in otherembodiments. The dose of 10 million cells was selected on the basis ofthe engraftment data from study 1, which had demonstrated, according toone embodiment, that the highest percentage engraftment of cellsoccurred when a smaller number was injected at each site in theperiinfarct border zone. (FIG. 33). Cells or placebo were injected underdirect visualization by open chest surgery performed 4 weeks after MI.The pigs were then followed for eight more weeks, to assess safety andefficacy. Thus, in one embodiment, injections are made in theperiinfarct border zone. In other embodiments, injections are madeoutside of said zone.

General anesthesia was induced in adult female Yucatan mini-pigs.Endotracheal intubation was then performed and anesthesia maintained.The mini-pigs were subjected to an anteroseptal MI by inflation of anangioplasty balloon in the mid-LAD to cause coronary occlusion for 2.5hours. Catheters were inserted via the left carotid artery. Afterreperfusion, during the same episode of general anesthesia, 4-6 rightventricular biopsies were obtained using a standard clinical cardiacbioptome introduced via the right internal jugular vein. The biopsieswere immediately placed into ice-cold cardioplegia solution (e.g., Ca⁺⁺and Mg⁺⁺ free PBS with 5% dextrose, mannitol 68.6 mmol/L, KCl 1.6mmol/L, NaHCO₃ 3.1 mmol/L and heparin) and cardiospheres or CDCs weregrown from these biopsy samples.

According to several embodiments of the invention, cardiac biopsyspecimens (10-40 mg) were minced, and subjected to collagenase IVdigestion. These explants were plated onto fibronectin-coated plasticplates with cardiac explant medium (IMDM (Invitrogen), 20% FBS, 1%penicillin-streptomycin, 1% L-glutamine, 0.1 mM 2-mercaptoethanol). Amonolayer of adherent cells grew out from the biopsy, which washarvested after 1-2 weeks. The harvested outgrowth was re-plated ontopoly-D-lysine coated wells. Under these conditions, within 3-5 days themajority of the cells gave rise to free-floating clusters of cells(e.g., cardiospheres). In a third phase, the adherent cells werediscarded, while the floating cardiospheres were collected and platedonce again onto fibronectin-coated cellware. The cardiospheres adheredand flattened to form a monolayer of cells referred to as CDCs, whichwere passaged as they became confluent. So called “secondarycardiospheres” were used in the in vivo experiments, meaning that anequivalent number of CDCs (e.g., about 10 million) were harvested andcounted, then plated back into poly-D-lysine coated wells where theyformed cardiospheres for a second time, which were injected into theanimals.

Cultured cells were transduced at the outgrowth stage with a lentiviralvector encoding the firefly luciferase gene, and further processed tocreate CDCs. Seven animals that had been subjected to the MI and RVbiopsy protocols, received intramyocardial injection of 0.5, 2.0 or 10million CDCs per injection site in intrainfarct, periinfarct(borderzone) or remote normal ventricular locations. (See FIG. 33) Otherdosages may be used according to other embodiments of the invention. Theanimals were sacrificed 24 hours later for assessment of cellengraftment.

Thirty-three pigs had general anesthesia induced a second time, 4 weeksafter MI, with the same drugs. Intramyocardial direct injection wasperformed by open chest surgery under sterile conditions. Sternotomy wasperformed and the pericardium opened to expose the heart. Twentyintramyocardial injection of either cardiospheres (0.5 million cellssuspended in 00.1 mL per injection), CDCs (0.5 million cells suspendedin 00.1 mL per injection) or placebo (00.1 mL of medium alone) wereperformed into the beating heart, using a 1 mL tuberculin syringe and 26gauge needle. The injections were spaced around the perimeter of themacroscopically visible infarct scar, approximately 1 cm from the grossborder. Other injection sites are used in according with otherembodiments of the invention. 11 pigs were allocated to receive placebo(vehicle alone), 8 allocated to receive cardiosphere injections, and 10allocated to receive CDC injections.

About 0.5M cells/site was administered per site in the periinfarct zonefor the study 2. Quantification of off-target expression at 24 hrsrevealed no measurable cells in liver, spleen or kidney, but 0.9% ofinjected CDCs could be detected in the lungs (see Table 4.) In severalembodiments, the percentage retained in the heart can be increased byiron-loading cells and applying an apical magnet, as described in PCTApplication No. PCT/US2010/054358, the disclosure of which is hereinincorporated by reference.

TABLE 4 Cell distribution in non-target tissues. Pig 1 Pig 2 Pig 3 Pig 4Pig 5 Pig 6 Pig 7 Lung 1.8% 0.3% 2.4% 0.6% 0.6% 0.3% 0.4% Liver — — — —— — — Spleen — — — — — — — Kidney — — — — — — —

On the basis of the 24 hour engraftment data, a dosage of 0.5M CDCs (oran equivalent cell number of cardiospheres) per site was selected, anddirect intramyocardial injections performed in 20 periinfarct sites,giving a total cell dosage of 10 million CDCs. FIG. 34A shows the LVEFdata derived from contrast ventriculography. LVEF at baseline wasequivalent in the three groups. FIG. 34B also demonstrates that, eightweeks post-injection, LVEF was significantly higher than placebo in bothof the cell treated groups, while there was no significant difference infinal LVEF between the CDC and the cardiosphere-treated groups. FIG. 34Cshows that the treatment effect (final minus baseline LVEF) wassignificantly higher than placebo in both of the cell treated groups.Thus, according to some embodiments of the invention, cardiac functionis improved when CDCs are administered. In some embodiments,cardiospheres improve cardiac function.

Echocardiographic measurement of LVEF yielded qualitatively similardifferences in final LVEF and delta LVEF measurements, though thedifferences between the groups were not statistically significant bythis modality (Table 5). Echocardiographic measurement did, however,demonstrate progressive ventricular dilatation in placebo and CDCgroups, which was attenuated in the cardiosphere-treated animals (FIG.35B and Table 5). Baseline systolic and diastolic LV volume measurementswere randomly lower in the CDC-treated animals (Table 5).

TABLE 5 Echocardiographic Indices Placebo CDC CSph p values: PlaceboPlacebo CDC (n = 9) (n = 9) (n = 5) ANOVA vs. CDC vs. CSph vs. CSphEjection fraction Baseline 43 ± 7  44 ± 12 43 ± 5  0.98* — — — Final 40± 7 47 ± 5 44 ± 5 0.07 — — — Treatment effect (delta)  −3 ± 11  +3 ± 10+1 ± 5 0.39 — — — systolic volume, mL Baseline 29.5 ± 4.8 24.7 ± 5.031.4 ± 3.9 0.04 0.04 0.49 0.02 Final  40.5 ± 11.8 34.7 ± 7.231.8 ± 5.60.21 — — — — Treatment effect (delta) +10.9 ± 13.2 +10.0 ± 6.2  +0.4 ±5.4 0.13 — — — Diastolic volume, mL Baseline 52.0 ± 9.4 44.2 ± 5.3 55.6± 9.8 0.04  0.054 0.44 0.02 Final  66.1 ± 12.9  65.0 ± 10.7 56.2 ± 7.80.27 — — — Treatment effect (delta) +14.0 ± 15.0 +20.8 ± 10.1 +0.7 ± 8.50.02 0.25 0.06 <0.01  Abbreviation: CSph, cardiospheres *BaselineEjection fraction did not exhibit homogeneity of variance betweengroups, so the Kruskal-Wallis test was performed instead of ANOVA

In addition, echocardiography revealed that final measurements of LVseptal wall thickness were increased in both of the cell-injected groupsrelative to placebo (FIG. 36B). The thickening fraction of the apicalseptum was also increased in cardiosphere-injected pigs. (FIG. 36C).Thus, in one embodiment cardiospheres are particularly efficacious.According to some embodiments, the invention provides improve morphologyand function in the infarct region with autologous CDC or cardiosphereinjections. In other embodiments, the invention provides improvemorphology and function in the infarct region with allogeneic CDC orcardiosphere injections.

Table 6 and FIG. 35A outline the results of LV pressure-volume loopanalysis. Most measurements were made at steady state. However oneimportant measurement, end-systolic elastance (Emax) was derived, bydefinition, as the slope of the end-systolic pressure-volumerelationship from the family of loops produced during balloon occlusionof the inferior vena cava. (FIG. 35A). Emax is a rigorousload-independent measure of contractility. Final Emax in thecardiosphere group was higher than in placebo-treated pigs, indicatingimproved left ventricular contractility in these animals (placebo1.03±0.29, CDC 1.66±0.45, cardiosphere 3.16±1.32 mmHg/mL. Kruskal-Wallisp=0.003. Placebo vs. CDC, p=NS; Placebo vs. cardiosphere, p=0.03;Cardiosphere vs. CDC, p=NS). Emax in the CDC group tended to increasebut was not significantly higher than in placebo-treated animals.

TABLE 6 Pressure-Volume Loop derived Indices Placebo CDC Cardiosphere (n= 11) (n = 10) (n = 7) ANOVAp Heart rate, bpm Baseline 121 ± 12 119 ± 12117 ± 17 0.72 Final 112 ± 17 109 ± 13 117 ± 19 0.65 Treatment effect  −9± 16  −9 ± 12  −1 ± 36 0.72** (delta) P_(max) mmHg Baseline  97.0 ± 11.586.9 ± 6.9  93.8 ± 14.6 0.13 Final  87.1 ± 11.4  88.4 ± 10.4 86.2 ± 8.10.90 Treatment effect  −9.9 ± 12.9  +1.5 ± 11.2  =7.9 ± 16.0 0.14(delta) LVEDP, mmHg Baseline 14.6 ± 3.1 14.5 ± 5.1 18.0 ± 5.3 0.20 Final12.6 ± 3.7 16.0 ± 6.2 12.1 ± 4.1 0.19 Treatment effect −1.9 ± 3.4 +1.5 ±4.4 −5.8 ± 1.7 0.01* (delta) dP/mt max Baseline 1967 ± 370 17709 ± 278 1784 ± 693 0.56 Final 1589 ± 446 1430 ± 370 1422 ± 333 0.58 Treatmenteffect −378 ± 462 −340 ± 280 −382 ± 876 0.93 (delta) dP/dt min Baseline−1984 ± 480  −1584 ± 346  −1784 ± 394  0.11 Final −1516 ± 291  −1527 ±357  −1546 ± 444  0.99 Treatment effect  468 ± 600  56 ± 501  247 ± 5200.25 (delta) tau, seconds Baseline 39.84 ± 4.87 38.93 ± 5.51 41.28 ±7.21 0.70 Final 41.25 ± 7.21 40.92 ± 4.57 41.88 ± 3.82 0.94 Treatmenteffect +1.41 ± 8.39 +1.98 ± 6.12 −0.03 ± 5.42 0.84 (delta)Abbreviations: P_(max), the maximum pressure generated by the leftventricle during the cardiac cycle; LVEDP, left ventricularend-diastolic pressure; dP/dt max, the maximum rate of rise of leftventricular pressure; tau, a measure of left ventricular relaxation.*For further details about post-hoc comparisons of delta LVDDP betweenthe three groups, please refer to FIG. 35C. **The delta heart ratevariable did not exhibit homogeneity of variance between groups, so theKruskal-Wallis test was performed instead of ANOVA.

Steady state hemodynamics showed few differences (Table 6) except for agreater fall in LV end-diastolic pressure in the cardiosphere-treatedgroup (FIG. 34C). Taken together with the lesser increase ofend-diastolic volume in this group, cardiosphere injected animalsexperience disproportionate benefit with regard to attenuation ofadverse ventricular remodeling relative to the other two groups (CDCs orplacebo).

Ventricular tachycardia was readily inducible by application ofprogrammed extra-stimuli in all animals prior to sacrifice, consistentwith previous reports. However, there were no deaths (sudden orotherwise) in either group after the immediate periprocedural period.Necropsy, with gross analysis as well as histology of heart, brain,kidney, lung, liver and spleen (Table 3) detected no tumors eight weeksafter intramyocardial injection of CDCs or cardiospheres.

Fluorescence immuno-histochemistry in the two animals with lacZ⁺ CDCs,revealed the presence of labeled cells 8 weeks after injection. FIGS.37A-37B shows two examples of islands of cardiomyocytes withlacZ-positive nuclei in the periinfarct zone, one from each animal thatreceived intramyocardial genetically-labeled CDCs. Thus, in oneembodiment, a proportion of injected autologous CDCs, or their progenywhich will also be labeled by this integrating vector, persist for 8weeks within the border zone of infarcted myocardium. In someembodiments, human cardiospheres have improved engraftment as comparedto human CDCs when injected into SCID mouse hearts under certainconditions and delivery mechanisms.

In several embodiments, direct surgical injection of autologouscardiospheres or CDCs effectively halts the deterioration in LVEF aftera large myocardial infarction, compared to a 7% absolute reduction inLVEF over eight weeks of observation in placebo-treated animals.Cardiospheres increased end-systolic elastance and attenuated theventricular dilatation associated with myocardial infarction. Althoughnot demonstrated in the study, CDC's may also exhibit similar effectsunder certain conditions.

According to several embodiments, short-term engraftment of about 8%regardless of injected cell dose in remote normal myocardium isfeasible. In some embodiments, in the infarct border zone, the percentsurvival at 24 hrs decreases progressively from ˜8% to <1% as dosageescalates. Thus, in one embodiment, the proportional engraftment ofinjected cells is improved by injection of lower cell doses at eachinjection site. In one embodiment, survival in the border zone may belimited by the tenuously-perfused, substrate limited, peri-infarctenvironment. In this scenario, while the absolute number of injectedcells able to survive remains about the same, percentage survival ofinjected cells is greater with injection of lower numbers of cells persite.

In some embodiments, engraftment rates are from 5%-10%, 10%-20%, 20-50%,50-75% and higher. In several embodiments, the following dosages of CDCsor cardiospheres per site are used: 0.1-0.2M, 0.2-0.3M, 0.3-0.4M,0.4-0.5M, 0.5-0.6M, 0.6-0.7M, 0.7-0.8M, 0.8-0.9M, 0.9-1.0M, 1.0-1.5M,1.5-2.0M, and higher, or overlapping ranges thereof. The followingnumber of injection sites are used in some embodiments: 1, 1-5, 5-10,10-15, 15-20, 20-25, 25-50, and more sites, and overlapping ragesthereof.

In some embodiments, the preservation of global LVEF in cell-treatedanimals, compared to the deterioration in the placebo group is providedwith intramyocardial injection. In several embodiments, cardiospheresare used to provide hemodynamic benefit and/or attenuation of adverseremodeling.

In some embodiments, CDCs comprise a natural mixture of progenitor andsupport cells expanded from myocardial biopsy specimens, withclonogenicity and multi-lineage potential. In one embodiment, CDCsprovide significant functional improvements even when engraftments ratesare low. In one embodiment, functional benefit involves indirect effectsto boost angiogenesis and cardiomyogenesis.

In some embodiments. regenerative cells described herein (e.g.,allogeneic or autologous cardiospheres and CDCs,) can be administered inprocedures where open chest surgery is performed (e.g., for othertherapy, such as implantation of cardiac devices). In some embodiments,less invasive methods of intramyocardial administration, such astrans-endocardial catheter-mediated delivery are used. In oneembodiment, regenerative cells are administered via an intracoronaryroute.

Example 13 Clinical Trials Using Autologous Cells

The preliminary safety and efficacy of autologous CDCs in patients withischemic left ventricular dysfunction and a recent myocardial infarctionhave been evaluated in a Phase I clinical trial (CADUCEUS, NCT00893360,which is incorporated by reference herein). Twenty-four patients (atleast 18 years of age with recent myocardial infarction and ischemicleft ventricular dysfunction) have or are scheduled to undergo a cardiacbiopsy to obtain tissue for generating the cell product. Autologouscardiosphere-derived stem cells, with a low dose of 12.5 million and ahigh dose of 25 million, are generated. Within 8 weeks of biopsy, and onaverage within 4 weeks, patients receive CDCs by intracoronary infusionin the infarct-related artery.

A total of 14 subjects have completed the 6 month follow up. In the 12.5million cell group, 4 subjects who received cells, 4 control subjectsand 1 intention to treat subject have completed their 6 month visit. Inthe 25 million cell group, 3 subjects who received cells, 1 controlsubject, and 1 intention-to-treat subject have completed their 6 monthvisit.

Magnetic resonance imaging (MRI) is being used to assess secondaryefficacy endpoints, including: changes in MRI-assessment of function inthe region which received CDC therapy; changes in MRI assessment ofinfarct size expressed in absolute value (grams) and as a percent of LVmass; changes in MRI assessment of perfusion in the region whichreceived CDC therapy; changes in MRI assessment of global LV function;and changes in MRI assessment of LV end-diastolic and end-systolicvolumes.

In terms of efficacy, the data sets are incomplete. Results to date havebeen summarized in tabular format below. Six-month follow-up MRIs havebeen completed for a subset of the study subjects: all 4 patientsrandomized to receive 12.5 M CDCs and 5 controls.

Although preliminary, the results to date are encouraging, in that therelative changes in infarct size by % LV infarcted are already showingdifferences between CDCs and controls. At baseline, i.e. before cellinfusion, infarct size was equivalent in the two groups (CDC 23.22% vs.Control 24.78%, p=0.84). By six months, those values trended in oppositedirections, CDCs getting smaller, as expected from the preclinical data(please refer to IND #13930 for details), and controls creeping upwards(CDC 17.14% vs. Control 28.97%, p=0.12). Comparison of the deltas(baseline minus 6 months) reveals a significant difference (CDC −6.08%vs. Control+4.2%, p=0.021).

The p values for absolute infarct mass are now nearly significant whencomparing infarct mass between the groups at 6 months, and for thecomparison of the change in infarct mass: Infarct Mass (g) Baseline: CDC26.24 g vs. Control 28.95 g, p=0.78 6 months: CDC 19.34 g vs. Control32.07 g, p=0.0684 Delta: CDC −6.9 g vs. Control+3.12 g, p=0.0676

Example 14 Protocols for Manufacturing of CDCs Cell Processing

A schematic showing one embodiment of cell processing is shown in FIG.38A-38F. In one embodiment, hearts will be collected from donors andtransported to the manufacturing facility. As described above the tissuewill go through three stages: 1) explants (EXP) stage: cell outgrowthfrom the tissue; 2) cardiospheres (CSP) stage: enrichment of cardiacstem cells by culturing; and 3) cardiosphere-derived cells (CDCs) stage:expansion of cells. A master cell bank (MCB) and working cell bank (WCB)of CDCs will be created as described below. In several embodiments,aliquots of the WCB equivalent to a single drug dosage will beformulated as the final composition to be administered.

Cell Banking System

In several embodiments, a cell banking system is provided comprising aplurality of cryogenically preserved, single use, populations of CDCsfor administration. In some embodiments, a master cell bank is provided.In some embodiments, a working cell bank is additionally provided (withCDCs that have undergone a greater number of passages). The disclosureherein details an example of a contemplated cell banking system.

In several embodiments, endomyocardial biopsies will be used to generatethe cell population for banking. In other embodiments, donor-qualityhearts will be dissected and biopsy-sized pieces are collected from theright ventricular septal wall, the atria, the apex, the rightventricular epicardium, or the left ventricular epicardium. Datademonstrate that CDCs can be produced using tissue from all regionsreliably (discussed above). In some embodiments, specimens will beprocessed immediately after collection, while in other embodiments, theywill be processed after up to about 6 days of storage in coldcardioplegia solution, and in still other embodiments aftercryopreservation and subsequent thawing. In some embodiments, a delay(from collection to processing) of about 3 days is the preferred maximaldelay. As discussed above, data demonstrate that the above mentionedtissue storage delays affect resultant CDC yield in an acceptablemanner.

In several embodiments, a master cell bank (MCB) will be created fromthe source material and a working cell bank (WCB) will be created fromthe MCB. The WCB will be aliquoted into single drug dosages which arethe final product. In some embodiments, the MCB will comprise cells thathave gone through the three stages of the culture process detailedabove: 1) the explants (EXP) stage: cell outgrowth from the tissue; 2)cardiospheres (CSP) stage: enrichment of cardiac stem cells; 3)cardiosphere-derived cells (CDCs) stage: expansion of cells. In severalembodiments, CDCs will be cryopreserved at 5 million cells permilliliter and stored in a liquid nitrogen tank located adjacent to themanufacturing facility. Other concentrations of CDCs may also be used,for example, a range of about 2-3 million cells per milliliter, about3-4 million cells per milliliter, about 4-5 million cells permilliliter, and overlapping ranges thereof. In some embodiments,concentrations above 5 million cells per milliliter will be used, as thehigher concentration help to ensure that, even if a portion of the cellsdie after cryopreservation, sufficient numbers are available in the dosefor use in therapy.

Genetic and phenotypic stability of the MCB will be assessed aftercryopreservation. Viability of the MCB will be assessed prior tocryopreservation and post-thaw.

The WCB will consist of CDCs from the MCB that undergo additionalpassages. Genetic and phenotypic stability of the WCB will be assessedprior to cryopreservation. Viability of the WCB will be assessed priorto cryopreservation and post-thaw. The final product consists of thosecells cryopreserved from the WCB. In several embodiments, a single WCBaliquot will be removed from the freezer for administration to thepatient. The post-thaw WCB aliquot represents the final product to bedelivered as the therapeutic. Stability of a cryopreserved product isdemonstrated above and will be confirmed for each lot of final productproduced. Efficacy of the final product will be verified in a mousemodel of myocardial infarction (see Examples for study plan).

As detailed above, a proof of concept banking system was generated. CDCswere passaged to P1 to create the MCB and subjected to testing assummarized below. A fraction of CDCs underwent further passaging to P6in order to generate a WCB. The yield presented for the WCB is thepotential yield extrapolated from the growth seen with the fraction ofcells expanded. CDCs will be taken up to P6 to generate the MCB and upto P12 for the WCB used in one embodiment. Testing to be employed on theMCB, WCB, and final product is described in detail above.

Reagents and Excipients

In several embodiments, several reagents used in the processing,culturing, cryopreservation, and administration of CDCs are animalorigin free (except for reagents such as serum and/or fibronectin, whichare of inherently animal origin). In some embodiments, the culturedcells are treated to remove any products of animal origin. In oneembodiment, fetal bovine serum is removed by washing the cells. In oneembodiment, the cells are washed with phosphate buffered saline. Inseveral embodiments, washing of cells removes fetal bovine serum suchthat the concentration remaining is less than about 0.05%. In oneembodiment, less than 0.0005 percent fetal bovine serum remains.

In several embodiments, dedicated protocols for collecting,manufacturing, and storing CDCs dedicated to allogeneic therapies areused, which are detailed above. While in some embodiments,endomyocardial biopsies may be used as a tissue source, in severalembodiments, transplant-quality hearts to generate allogeneic CDCs. Insome embodiments, a master cell bank is generated and in someembodiments, a working cell bank is generated. Thus, in someembodiments, allogeneic CDCs are cryopreserved prior to administrationto the patients. Depending on whether CDCs are cryopreserved (certainallogeneic embodiments) or administered to a patient withoutcryopreservation (certain autologous and certain allogeneic embodiments)the final excipients in the administered composition will vary. Thesingle excipient in cryopreserved products is CRYOSTOR® CS5, acryoprotectant solution. CRYOSTOR®CS5 is GMP manufactured, made with USPgrade components, serum-free, protein-free, and animal-origin free.

Example 15 Clinical Trials Using Allogeneic Cells

Cardiosphere-derived cells were used in patients with left ventriculardysfunction and a recent myocardial infarction with product deliveryoccurring by intracoronary infusion via an over-the-wire ballooncatheter. The following sets forth an example of a contemplated trialusing allogeneic cells.

In several embodiments, allogeneic cells would be used in patients withdamaged heart tissue. Damaged heart tissue includes heart tissue withsub-optimal function. In one embodiment, the patients have one or moreof the following: left ventricular dysfunction, prior myocardialinfarction and LVAD placement. Patient may be receiving LVAD placementeither as a bridge to transplantation or as destination therapy. In oneembodiment, the patients will be undergoing LVAD placementsimultaneously with the administration of allogeneic cells. In someembodiments, the use of allogeneic cells as an adjunct to anothertherapy (e.g., LVAD) will work synergistically with said therapy. In oneembodiment, the delivery of regenerative cells (autologous orallogeneic) according to several embodiments herein is used prior,during and/or after heart transplant surgery.

Several delivery approaches may be used. For example, intramyocardialinjection using a standard needle and syringe and an epicardial approachduring LVAD placement is used in some embodiments. In one embodiment,intramyocardial injection using a dosage of about 5-15 million (e.g., 10million) CDCs is used. Other agents, such as preservatives, carriers andrecipients may be delivered with the cells, including but not limited toDMSO. In one embodiment, 2 mLs of solution containing 5% DMSO (100 μLsof DMSO) will be administered by intramyocardial injection. In oneembodiment, patients will receive a single dose of 10 million CDCsdelivered with a standard needle and syringe. Each injection willconsist of 100 microliters containing 0.5 million CDCs. A total of 20epicardial injections will be made during LVAD placement. Dosages,number of injections an injection sites will vary in other embodiments.

In one embodiment, patients treated with allogeneic cells (or autologouscells) according to several embodiments described herein, will show oneor more of the following improvements: (i) weaning success during thestudy period such that LVAD removal is feasible, (ii) improved LVAD RPMat which aortic valve opening in every cardiac cycle is seen bytransthoracic echo, the corresponding LV ejection fraction, wallthickness, and mass, (iii) improved perfusion and coronary blood flowreserve, and (iv) improved walk distance/times and other exercise testparameters.

In some embodiments, patients will be weaned of LVAD (or other therapy)through the use of regenerative cell therapy. In one embodiment, weaningwill take place over the course of 1-3 months. In one embodiment, on aweekly basis, patients will undergo transthoracic echo to assess LVfunction and aortic valve opening and to assure aortic valve closure andadequate volumetric pump flow rate during RPM adjustment. LVAD flow willbe adjusted downward 100-500 RPM per week until the target level of 9000RPM is reached (baseline settings are typically 9500-10000 RPM). Pumpflow rate and RPM will be recorded before and after pump adjustment.During this time, INR will be maintained between 1.5 and 2.5 to minimizethe risk of thrombus formation. During these weekly visits, the 6-minutewalk test and cardiopulmonary exercise test (as patients are able) willbe performed. LVAD explantation will be considered if patients are ableto perform cardiopulmonary exercise tests.

In one embodiment, once the LVAD flow has been adjusted to 9000 RPM,patients will be admitted to the hospital to undergo a rapid wean. Anechocardiogram will be performed at 9000 RPM to measure baseline EF, LVdimensions and Systolic velocity (Sm) of the basal segments (septal andlateral wall) by Tissue Doppler. The device will then be graduallyadjusted to 6000 RPM with reductions of 1000 RPM occurring every 4-6hours. A BNP value will also be obtained every 4-6 hours. If the patientreports symptoms or there is an increase in BNP to a value >1000 pg/mL,the rapid weaning attempt will be terminated, and the LVAD speed will beadjusted back to 9000 RPM. 6000 RPM has been determined to be a safespeed for device function, without increased risk for thrombosis orbackflow from the aorta to the LV. The device will remain at this speedfor 16-24 hours. If the patient successfully completes the 16-24 hourperiod at 6000 RPM (asymptomatic, BNP <1000 pg/mL), an echocardiogram tomeasure changes in EF, LV dimensions and Sm will follow. If EF>45%,right heart catheterization will follow, with the device at 6000 RPM, inorder to measure PCWP. Finally, a symptom limited CPX will follow, withthe device set at 6000 RPM. If the patient satisfies the explantationcriteria (see below) the device will be set back to the optimal RPMlevel specified before the initiation of the weaning protocol, untilexplantation occurs. In some embodiments, LVAD explantation will beperformed if the following criteria are met: (i) a LVEF >45%, (ii) LVEDD<55 mm, (iii) Sm >8 cm/sec, (iv) a change in these three parameters of<10% at 6000 RPM compared to 9000 RPM, (v) LVEDP <12 mm (vi) Hg or PCWP<12 mm Hg, (vii) resting cardiac index >2.8 L/min/m², (viii) exerciseVO2 max >16 mL/kg/min at 6000 RPM, and (ix) adequate right ventricularfunction (under minimal LVAD support at 6000 rpm), assessed byechocardiography and right heart catheterization. In one embodiment, atleast one of the criteria is met before explanation. Thus, according toseveral embodiments of the invention, administration of regenerativecells advantageously permits LVAD explanation.

Example 16 Efficacy of CDCs for Myocardial Repair Versus AlternativeStem Cell Types

As discussed above, several embodiments of the present inventioncomprise use of CDCs for regeneration and/or repair of damaged cardiactissue. Use of heart-derived cells e.g., cardiospheres, CDCs) forregenerative cardiology is but one of several approaches presently beingemployed in the pursuit of cardiac cell therapy. Multiple extra-cardiaccell types, including bone marrow mononuclear cells (BM-MNCs), bonemarrow-derived mesenchymal stem cells (BM-MSCs), adipose tissue-derivedmesenchymal stem cells (AD-MSCs), endothelial progenitor cells, andmyoblasts are under investigation for use in regeneration of the damagedheart. Moreover, even within heart-derived cells, there are multipleapproaches being investigated. For example, the CDCs disclosed herein(mixture of stromal, mesenchymal and progenitor cells) are used inseveral embodiments to effect cardiac repair and/or regeneration. Whilein some embodiments, selection is performed, in several embodiments, theCDCs are not selected for, or enriched based on expression of anyparticular markers. One alternative approach is to purify the c-kit⁺subpopulation from mixed heart-derived cells. Thus, the present studywas performed to compare the efficacies of several of these various celltypes in repairing and/or regenerating cardiac tissue by direct and/orindirect mechanisms.

Methods Cell Sources

Human CDCs were obtained and expanded as described above. Human BM-MSCsand BM-MNCs were purchased from Lonza (Walkersville, Md.). Human AD-MSCswere purchased from Invitrogen (Carlsbad, Calif.). These cells werefreshly-isolated from healthy donors. The c-kit⁺ stem cell subpopulationwas purified from the expanded CDC population using a CELLection PanMouse IgG Kit and a Dynal Magnetic Particle Concentrator-15(Invitrogen).

For confirmatory rat studies, four-month-old Wistar Kyoto rats were usedto expand CDCs, BM-MSCs, and AD-MSCs. BM-MNCs were also collected fromthe same rats by gradient centrifugation. Freshly-collected BM-MNCs andtwice-passaged CDCs, BM-MSCs, and AD-MSCs were used for the ratexperiments discussed below.

Unless otherwise noted, IMDM basic medium (Gibco) supplemented with 10%FBS (Hyclone) and 20 mg/ml gentamycin was used to culture all celllines.

Flow Cytometry

Characterization of CDCs, BM-MSCs, AD-MSCs, and BM-MNCs was evaluated byflow cytometry using established methods. Briefly, cells were incubatedwith FITC or PE-conjugated antibodies against CD29, CD31, CD34, CD45,CD90, CD105, c-kit, and CD133 (eBioscience) for 30 minutes.Isotype-identical antibodies served as negative control. Quantitativeanalysis was performed using a FACSCalibur flow cytometer with CellQuestsoftware (BD Biosciences).

ELISA

For evaluation of growth factor production, cells were seeded in 24-wellculture plates at densities of 1×10⁶/ml (BM-MNCs) or 1×10⁵/ml (all othercell types) in FBS-free IMDM media (all cell types) for 3 days.Supernatants were collected and the concentrations of angiopoietin-2,bFGF, HGF, IGF-1, PDGF, SDF-1, and VEGF were measured with human ELISAkits (R&D Systems Inc.), according to the manufacturer's instructions.For evaluation of cytokine production by cultured rat cells,concentrations of HGF (B-Bridge International, Inc.), IGF-1, and VEGF(R&D Systems Inc.) were measured in the supernatants after 3 days ofculture.

To compare the production of growth factors from the purified c-kit⁺subpopulation and unsorted CDCs, cells (5×10⁴/ml) were seeded in 24-wellculture plates and cultured for 2 days under 20% O₂. Growth factors inconditioned media were measured by ELISA as described above.

Immunostaining

To determine myogenic differentiation in vitro, cells were seeded onfibronectin-coated 4-chamber culture slides. After 7 days of culture,cells were fixed, blocked with goat serum for 30 minutes, and thenincubated with mouse anti-human troponin T antibody (R&D Systems Inc.)for human cells or with goat anti-rat troponin T antibody for rat cells.After 1 hour incubation at room temperature, culture slides were washedand then incubated with a PE-conjugated secondary antibody. Cell nucleiwere stained with DAPI. Cardiomyogenic differentiation was quantified bycounting positively-stained cells.

In Vitro Angiogenesis Assay

Angiogenic potency was assayed by tube formation using a kit (ChemiconInt.), according to the manufacturer's instructions. Briefly, cells wereseeded on ECMatrix™-coated 96-well plates at a density of 2×10⁵ cells(BM-MNCs) or 2×10⁴ cells (all other cell types) per well. HUVEC cellswere included as positive controls. After 6 hours, tube formation wasimaged. The total tube length was then measured with Image-Pro Plussoftware (version 5.1.2, Media Cybernetics Inc., Carlsbad, Calif.).

TUNEL Assay

To quantify the resistance to oxidative stress in vitro, cells wereseeded on fibronectin-coated 4-chamber culture slides. After 24 hours ofculture, cells were cultured with or without the addition of 100 μM H₂O₂to the medium for another 24 hours. Cells were fixed, and apoptoticcells were detected by TUNEL assay using the In Situ Cell DeathDetection Kit (Roche Diagnostics, Mannheim, Germany), according to themanufacturer's instructions. Cell nuclei were stained with DAPI;apoptotic cells were counted by TUNEL-positive nuclei.

Myocardial Infarction Model and Cell Implantation

Acute myocardial infarction was created in male SCID-beige mice (10-12weeks old), as described above. Cells were injected at four points inthe infarct border zone with a total of 40 μA of one of the following:phosphate-buffered saline (Control, n=8), 1×10⁵ CDCs (CDCs, n=20), 1×10⁵BM-MSCs (n=20), 1×10⁵ AD-MSCs (n=20), 1×10⁶ BM-MNCs (high BM-MNCs,n=11), or 1×10⁵ BM-MNCs (low BM-MNCs, n=9). Two dosages were studiedwith the BM-MNCs, including one with 10-fold more cells than in thecomparator groups, because MNCs are smaller than the other cell types,thus the higher dose avoids experimental bias against this cell type interms of total transplanted cell mass.

C-kit-selected and non-selected CDCs were compared in a separate studyby injecting 1×10⁵ purified c-kit⁺ cells (c-kit⁺, n=16) and 1×10⁵unsorted CDCs (unsorted, n=11) into the infarcted hearts of SCID mice,using the methods described above.

Echocardiography

Mice underwent echocardiography 3 hours (baseline) and 3 weeks aftersurgery using Vevo 770™ Imaging System (VISUALSONICS™, Toronto, Canada).After the induction of light general anesthesia, the hearts were imagedtwo-dimensionally in long-axis views at the level of the greatest leftventricular (LV) diameter. LV end diastolic volume, LV end systolicvolume, and LV ejection fraction (LVEF) were measured with VisualSonicsV1.3.8 software from 2D long-axis views taken through the infarctedarea. Blinded reading of echos was conducted independently by twoexperienced echocardiographers (K. M. and J. T.). The results correlatedwell (FIGS. 39A-39B), so the averages of the two readings for LVEF ineach mouse were used for statistical analysis.

Histology

Mice were sacrificed 3 weeks after treatment. Hearts were sectioned in 5μm sections and fixed with 4% paraformaldehyde. The engraftment ofimplanted human cells was identified by immunostaining for human nuclearantigen (HNA; Chemicon Int.). To measure cell engraftment, 10 images ofthe infarct and border zones were selected randomly from each animal. Toquantify the apoptotic cells in the heart, slides were fixed andapoptotic cells were detected by TUNEL assay as described above. Thedifferentiation of implanted human cells into cardiomyocytes in theinfarcted hearts of SCID mice was identified by immunostaining withmonoclonal antibodies against human specific α-sarcomeric actin (Sigma),as described above. For morphometric analysis, animals in each groupwere euthanized at 3 weeks (after cardiac function assessment) and thehearts were harvested and frozen in OCT compound. Sections every 100 μm(5 μm thick) were prepared. Masson's trichrome staining was performed asper manufacturer's instructions (HT15 Trichrome Staining (Masson) Kit;Sigma). Images were acquired with a PathScan Enabler IV slide scanner(Advanced Imaging Concepts, Princeton, N.J.). From the Masson'strichrome-stained images, morphometric parameters including infarct wallthickness and infarct perimeter were measured in each section with NIHImageJ software.

Statistical Analysis

All results are presented as mean±standard deviation (SD) except asnoted. Statistical significance was determined by one-way ANOVA followedby LSD post hoc test (Dr. SPSS II, Chicago, Ill.). Three outliers (2from CDC group and 1 from AD-MSC group), which differed from the meansof each group by >2 SDs, were omitted, and differences were consideredstatistically significant when p<0.05.

Results Characterization of Cell Phenotypes

Unlike BM-MNCs, which grow in suspension as small round cells, all othercell types studied (CDCs, BM-MSCs, and AD-MSCs) typically grow asadherent monolayers (compare FIGS. 40A-40C to 40D). Flow cytometrydistinguished BM-MNCs from other cell types by the predominantexpression of pan-hematopoietic marker CD45 (74.7%), as compared to <1%in CDCs, BM-MSCs, and AD-MSCs (FIG. 40E).

Conversely, >99% of CDCs, BM-MSCs, and AD-MSCs expressed CD105, a TGF-βreceptor subunit commonly associated with MSCs. However, these threecell types can be distinguished by CD90: >99% of BM-MSCs and 85% ofAD-MSCs expressed CD90, but only 18% of CDCs expressed this marker. CD90(well-known as Thy-1) was originally discovered as a thymocyte antigen.In humans, Thy-1 is also expressed by endothelial cells, smooth musclecells, a subset of CD34⁺ bone marrow cells, and umbilical cord blood,fibroblasts, and fetal liver-derived hematopoietic cells. CD90 is widelyused as a marker of a variety of stem cells, e.g. MSCs, hepatic stemcells, keratinocyte stem cells, putative endometrial progenitor/stemcells, and hematopoietic stem cells. Thus, in some embodiments, CDCscontain a minority of fibroblast and/or weakly-committed hematopoieticcells, which is in contrast to the dominance of such populations in thecells of bone marrow and adipose origins. In some embodiments, CD90expression in CDCs marks the cardiac mesenchymal subpopulation. In stilladditional embodiments, at least a portion of CDCs do not express CD90.

In Vitro Secretion of Growth Factors

As discussed above, paracrine mechanisms are at least partiallyresponsible, in some embodiments, for the repair and or regeneration ofcardiac tissue. Production of six growth factors (angiopoietin-2, bFGF,HGF, IGF-1, SDF-1, and VEGF) by the various cell types was compared.Compared to the other cell types, CDCs were unique in their ability tosecrete large amounts of all growth factors studied (see FIGS. 41A-41F).In contrast to CDCs, the other cell types failed to express comparablelevels of one or more growth factors:

-   BM-MNCs produced little VEGF and SDF-1; BM-MSCs secreted little    IGF-1 and bFGF; and AD-MSCs were not rich sources of HGF and SDF-1.    FIGS. 41G-41J depicts schematically the secretion of each of the six    studied cytokines in each given cell type, as wheel-and-spoke    diagrams in which the length of each spoke is proportional to the    growth factor concentration in conditioned media. The symmetrical    starburst pattern highlights the uniquely well-balanced paracrine    profile of CDCs.

In order to ensure that these findings did not reflect donor-specificidiosyncrasies (commercially purchased cell types were from differentdonors), growth factor secretion by the various cell types derived fromindividual rats were also compared. Correlating with the results above,higher levels of VEGF, IGF-1, and HGF (FIGS. 42A, 42B, and 42C,respectively) were detected in media conditioned by rat CDCs as comparedto rat BM-MSCs, AD-MSCs, and BM-MNCs, all collected from the sameanimals. In several embodiments, it is this unexpectedly balancedsecretion of various paracrine factors that provides, at least in part,the therapeutic benefits of the CDCs. In some embodiments, thewell-balanced release of growth factors by CDCs, which are acting aslocalized production factories post-administration, favors enhancedmyocardial repair through paracrine mechanisms after implantation intothe heart. However, in some embodiments, the generation of one or moreof these factors that is greater than the amount generated by the othercell types is responsible, at least in part, for the enhanced efficacyof CDCs. For example, in some embodiments, the amount of VEGF secretedby CDCs is greater than that of the other cell types, and lead (at leastin part) to the enhanced therapeutic effect seen post-CDCadministration. In still additional embodiments, generation of one ormore of these factors (or other factors disclosed herein) functions inconcert with direct mechanisms (e.g., engraftment of the CDCsthemselves) to effect cardiac repair and or regeneration.

Tube Formation

The angiogenic ability of the various cell types was quantified using anin vitro tube-forming assay. All cell types showed the ability to formcapillary-like networks on matrigel within 6 hours (FIG. 43C, upperleft, upper right, and lower left panels), with the exception of BM-MNCs(FIG. 43C, lower right panel). Quantitative analysis showed that themean tube length of the capillary-like networks was greater in CDCs thanin the other cell types (p<0.05, FIG. 43D). Thus, in severalembodiments, administration of CDCs yields a greater angiogenic effectin the target tissue. In some embodiments, this effect yields formationof longer vessels, thereby maintaining or improving blood supply tocardiac tissue. In some embodiments, the longer vessels formed allowblood supply to bypass a damaged region. In some embodiments, theincreased angiogenic effect yields a more dense or more branched networkof vessels (e.g., capillaries), which thereby maintains or improvesregionalized blood supply to cardiac tissue (e.g., a certain region ofcardiac tissue is more thoroughly perfused). Combinations of increasedlength and increased density result in some embodiments. This increasein the vascular infrastructure results in greater capacity fordistribution of blood to the cardiac tissue. As a result, in severalembodiments, the increased angiogenic effect of CDCs mediates thefunctional recovery and or anatomical repair and/or regeneration ofcardiac tissue through one or more of increased blood flow, increasedability to distribute paracine factors, increased oxygen delivery to themyocardium, and combinations thereof.

Resistance to oxidative stress

According to several embodiments disclosed herein, the short-termsurvival and/or engraftment of administered cells plays a role in therepair or regeneration of damaged cardiac tissue. Survival of theadministered cells despite the rigors of transplantation proceduresenables CDCs, in some embodiments, to provide greater therapeuticbenefits. For example, enhanced cell resilience to oxidative stressfavors both transplanted cell engraftment and resultant functionalbenefit. Sensitivity to oxidative stress was assessed by exposing cellsto H₂O₂, a powerful oxidant. After 24 hours of exposure to 100 μM H₂O₂,the number of apoptotic cells tended to be lower in human CDCs ascompared to human BM-MNCs (p=0.067, FIG. 44B), but there was nosignificant difference among CDCs, BM-MSCs, and AD-MSCs. In rat cells,higher apoptosis was observed in BM-MNCs compared to any of the otherthree cell types after H₂O₂ (p<0.05, FIG. 45B). Taken together, thesedata highlight a relative deficiency of BM-MNCs in terms of resistanceto oxidative stress. These data also suggest, that, in some embodiments,CDCs show resistance to oxidative stressors. As a result, the increasedresistance may enable CDCs (or cardiospheres) to survive longer,function more robustly, and/or engraft to a greater degree than otherstem cell types. In combination with other characteristics (e.g.,paracrine profile) of the CDCs generated according the methods disclosedherein, a greater therapeutic efficacy is realized, in severalembodiments.

Cardiomyogenic differentiation

As discussed above, in several embodiments CDCs are suitable fordifferentiation into cardiomyocytes. In some embodiments, this accountsfor (at least a portion of) the repair or regeneration of damaged ordiseased cardiac tissue via a direct mechanism. The ability of thevarious cell types to undergo spontaneous cardiomyogenic differentiationin vitro was assessed by immunostaining for cardiac-specific troponin T.Many human CDCs expressed troponin T (see, e.g., FIGS. 43A and 43B), incontrast to human BM-MSCs, AD-MSCs, or BM-MNCs, few of which werepositive for troponin T. Quantitative analysis showed that ˜9% of CDCsexpressed troponin T, while <1% did so in the other cell types (FIG.43B). Similar findings were observed using rat CDCs, BM-MSCs, AD-MSCs,and BM-MNCs, all collected from the same animals (FIGS. 46A-46B). Thus,in some embodiments, the direct differentiation of CDCs (or othercardiac stem cells such as cardiospheres) into cardiac cells serves as aprimary mechanism for the repair and/or regeneration of functionalcardiac tissue. In some embodiments, the direct differentiation is a asa complementary mechanism, working in concert with the paracrine effectsdiscussed herein.

Cell engraftment and in vivo differentiation

As disclosed above, the methods and compositions disclosed herein yielda positive correlation between long-term cell engraftment and functionalbenefit (e.g., functional and/or anatomical cardiac repair and/orrecovery). Engraftment and differentiation of human cells 3 weeks afterdirect intramyocardial injection into the infarcted hearts of SCID micewas evaluated. Histology revealed expression of α-sarcomeric actin (αSA)in some of the surviving progeny of human CDCs (positive for humannuclear antigen [HNA]; FIG. 47A), confirming the cardiomyogenicdifferentiation in vivo. In contrast, human cells positive forα-sarcomeric actin were observed rarely and inconsistently in miceinjected with BM-MSCs, AD-MSCs, and BM-MNCs (data not shown).Quantitative image analysis confirmed that the engraftment (e.g., thenumbers of HNA⁺ cells) was greater in mice implanted with human CDCsthan with comparator cells (p<0.05, FIG. 47B). In addition, the numbersof cardiomyocytes derived from the transplanted cells (HNA₊/αSA⁺) weregreater in mice implanted with human CDCs than with any of the othercell types (p<0.01, FIG. 47C). In several embodiments, the increasedcardiomyogenic differentiation attributable to CDCs is responsible forthe greater therapeutic efficacy of CDCs. In some embodiments, theincreased engraftment alone is responsible for improved efficacy, notonly because a greater number of administered CDCs are retained in thetarget cardiac tissue, but because there is an associated greaterincrease of paracrine factor production. In some embodiments, thegreater degreed of cardiomyogenic differentiation is responsible for thegreater efficacy (e.g., direct mechanisms of repair are dominant). Inseveral embodiments, these effects of CDCs (increased engraftment,increased cardiac differentiation, paracrine effects) function inconcert to for a multi-pronged mechanism of cardiac tissue repair and/orregeneration.

Cell apoptosis

In addition to tissue regeneration, tissue preservation may be asalutary component of cell therapy for acute myocardial infarction. Toevaluate this possibility, apoptotic nuclei in the infarcted region ofcontrol mice and mice injected with each of the comparator cell typeswere evaluated. TUNEL staining revealed apoptotic nuclei in theinfarcted hearts 3 weeks after treatment (FIGS. 48A-48B). Given thetime-point, it is likely that the acute phase of cell death due toischemia may have already resolved. Thus, the apoptotic nuclei may be areflection of long-term remodeling and heart failure. The total numberof apoptotic cells in the infarct and peri-infarct area was counted. Thehearts of mice implanted with CDCs exhibited fewer TUNEL-positive cells,compared to all other cell-treated groups (p<0.05, FIG. 48C). Asdiscussed above, the greater production of pro-angiogenic andanti-apoptotic factors by CDCs may be responsible, at least in part, forthe reduced apoptosis of CDCs. Moreover, in some embodiments, thereduced apoptosis may be responsible for the increasedengraftment/survival of CDCs. In some embodiments, however, theincreased engraftment may be responsible for the reduced apoptosis(e.g., the engrafted cells are in a preferred environment for survivalas compared to loosely attached or non-engrafted cells). Regardless ofthe temporal order of these mechanisms, the reduced amount of apoptosisallows for one or more of increased CDC survival, increasedcardiomyogenic differentiation, and increased paracrine factorproduction, which in turn result in one or more of improved cardiacanatomy (e.g., reduced infarct size) or improved cardiac function (e.g.,increased LVEF). Moreover, in several embodiments the reduction inapoptosis is realized not only in the acute phase of cell death (e.g.,at short time periods after an ischemic event), but also in thelong-term (e.g., ameliorating long-term remodeling and/or heartfailure).

Cardiac function

Clinically, one of the most meaningful endpoints of cardiac cell therapyis the ability to produce functional benefit after transplantation intothe injured heart. Echocardiography was used to measure cardiacfunction, and all images were interpreted blindly and independently bytwo experienced sonographers (see FIGS. 39A-39B). FIG. 49 summarizes theresults. The LVEF at baseline (i.e., two hours post-infarction) wascomparable among all groups. This indicates similar ischemic injuryamong the groups. Among the various treatments, the implantation of CDCsresulted in the greatest LEVF at 3 weeks (p=0.038 vs. BM-MSC; p=0.002vs. AD-MSC; p=0.002 vs. high BM-MNC; p=0.001 vs. low BM-MNC group; andp<0.001 vs. Control). BM-MSCs also improved cardiac function (p=0.009vs. Control) and AD-MSCs tended to improve function (p=0.073 vs.Control), while the other cell types, although higher on average thancontrols, had no statistically significant functional benefit. Thus, insome embodiments, administration of stem cells prevents the decline incardiac function that results from cardiac injury absent any therapeuticintervention. Advantageously, the administration of cardiac stem cells(e.g., CDCs) not only prevents this decline in function, but yieldsimproved function over time. In several embodiments function is improvedat least about 5% over a baseline function (e.g., function afterinjury). In several embodiments function is improved at least about 10%over a baseline function. In still additional embodiments, greaterimprovements in function are realized.

Ventricular remodeling

Potentiating the functional benefits of cell therapy is attenuation ofadverse ventricular remodeling. To evaluate this effect, themorphological consequences of transplantation of the various cell typeson myocardial infarct size and wall thinning were evaluated. Heartmorphometry at 3 weeks showed severe LV chamber dilatation and infarctwall thinning in the control hearts (FIGS. 50A-50F). In contrast, allthe cell-treated groups exhibited attenuated LV remodeling. Compared tocontrol, the implantation of any type of human cells decreasesfractional infarct perimeter and, conversely, increases the minimalinfarct wall thickness, 3 weeks after treatment (p<0.05 vs. Controlgroup, FIGS. 50G-50H). Despite the positive benefits due toadministration of any cell type, the protective effect was greatest inthe CDC-treated hearts. CDC-treated hearts had thicker infarcted walls(FIG. 50G; p<0.01), but a smaller fractional infarct perimeter (FIG.50H; p<0.05) as compared to any of the other cell-treated groups. Thus,in addition to the above-discussed improved function, the administrationof CDCs, in several embodiments, also improves or mitigates theremodeling that occurs after an injury to the heart (e.g., an ischemicevent). In some embodiments, infarct size is reduced by 10% to about 50%as compared to infarct size in an untreated subject. In someembodiments, infarct size is reduced 2-fold, 3-fold, 5-fold or greater.In some embodiments, functional cardiac tissue mass is increased (e.g.,increased wall thickness). In several embodiments, the increases areabout 2-fold, 3-fold, 5-fold or greater than an untreated control. Insome embodiments, the combination of reduced infarct (or other damage)size and increased functional mass provide a synergistic increase incardiac function.

Unsorted CDCs versus the c-kit⁺-purified cell subpopulation

Having established that CDC populations that are unselected for anyparticular marker were the most efficacious cell type among thosestudied, a comparison of such CDCs was made against purified c-kit⁺cells. Unsorted CDCs were compared to equal numbers of c-kit⁺ stem cellspurified from CDCs by magnetic cell sorting. Purified c-kit⁺ cells weredetermined to be inferior to unsorted CDCs in terms of functionalbenefit after transplantation into the infarcted heart, although theydid outperform vehicle-injected controls (FIG. 51A). The sortingprocedure did not itself compromise cell functional efficacy, as CDCssorted for CD105 (expressed by >99% of CDCs) exhibited an LVEFcomparable to that of unsorted CDCs (data not shown). The c-kit antibodyused for purification is known to interfere only minimally with ligandbinding, receptor phosphorylation, and internalization inc-kit-expressing cell lines. Also, magnetic-activated cell sorting formast cells using this c-kit antibody neither induced histamine releasenor did it impair the ability of cells to release histamine whenstimulated. The therapeutic superiority of the CDCs versus the purifiedc-kit⁺ subpopulation suggests that, in some embodiments, the mixed CDCpopulation (e.g., stromal, mesenchymal, and c-kit⁺ cells) function inconcert to enhance overall paracrine potency, direct repair mechanisms,and in turn functional and anatomical benefits.

To investigate one potential mechanism for the functional superiority ofCDCs, production of a variety of paracrine factors in conditioned mediafrom sorted and unsorted cells was evaluated. Indeed, unsorted CDCsproduced higher amounts of paracrine factors in vitro as compared topurified c-kit⁺ cells (FIGS. 51B-51E). As discussed above, in someembodiments, the well-balanced release of growth factors by CDCspromotes enhanced myocardial repair through paracrine mechanisms afterimplantation into the heart. In some embodiments, the combination of thebalanced profile and the overall greater amount of production isresponsible for the increased efficacy of cell therapy with unsortedCDCs. In some embodiments, it is the overall greater amount ofproduction which is responsible for the increased efficacy of celltherapy with unsorted CDCs. In still additional embodiments, generationof one or more of these factors (or other factors disclosed herein)functions in concert with direct mechanisms (e.g., engraftment of theCDCs themselves) to effect cardiac repair and or regeneration.

1. A method for the reduction of teratoma formation following thedelivery of non-self cells to a first subject, comprising: delivering toa first subject a population of regenerative cells, wherein saidregenerative cells are isolated from a tissue source harvested from asecond subject, wherein said regenerative cells express one or morefactors that reduce teratoma formation, wherein said at least a portionof said regenerative cells engraft into a target tissue of said firstsubject after delivery to said subject, wherein said engraftmentpersists for a time period ranging from about 1 week to about 6 weeks,wherein during said period of engraftment at least a portion of saidregenerative cells are destroyed by the immune system of said firstsubject, and wherein said engraftment of a portion of said regenerativecells, said period of engraftment, and said destruction of saidregenerative cells reduces teratoma formation.
 2. The method of claim 1,wherein the reduction in teratoma formation is in comparison to teratomaformation after delivery of embryonic cells to a subject.
 3. The methodof claim 1, wherein said second subject is an adult.
 4. The method ofclaim 1, wherein during said period of engraftment, said regenerativecells induce endogenous cells to express one or more factors that reduceteratoma formation.
 5. The method of claim 1, wherein delivery of saidregenerative cells is for the purpose of repairing a damaged or diseasedtissue of said first subject.
 6. The method of claim 5, wherein saiddamaged or diseased tissue of said first subject comprises damaged ordiseased cardiac tissue and wherein said population of regenerativecells comprises cardiac stem cells.
 7. The method of claim 6, whereinsaid cardiac stem cells are selected from the group consisting ofcardiospheres, cardiosphere-derived cells, and a subsequent generationof cardiospheres.
 8. The method of claim 1, wherein the regenerativecells express one or more stem cell markers selected from the groupconsisting of c-kit, CD90, and sca-1.
 9. The method of claim 1, whereinthe regenerative cells express one or more endothelial cell markersselected from the group consisting of KDR, flk-1, CD31, von Willebrandfactor, Ve-cadherin, and smooth muscle alpha actin.
 10. The method ofclaim 1, wherein the regenerative cells express one or more of said stemcell markers or one or more of said endothelial cell markers, but arenot selected for based on the expression of said one or more expressedmarkers.
 11. The method of claim 1, wherein said isolated regenerativecells are expanded in culture prior to delivery.
 12. The method of claim11, wherein said isolated regenerative cells generate teratoma-reducingfactors in culture.
 13. The method of claim 12, further comprisingisolating said teratoma-reducing factors from said culture.
 14. Themethod of claim 13, further comprising delivering said isolatedteratoma-reducing factors from said culture to said first subject. 15.The method of claim 14, wherein delivery of said isolatedteratoma-reducing factors is prior to, concurrently with, or afterdelivery of said regenerative cells.
 16. The method of claim 15, whereindelivery of said isolated teratoma-reducing factors is at multiple timepoints throughout the period of engraftment of said regenerative cells.17. The method of claim 1, wherein expression of said factors comprisescell-surface expression.
 18. The method of claim 1, wherein expressionof said factors comprises release of said factors from the cells.
 19. Amethod of treating a first subject having damaged cardiac tissue withallogeneic cells from a second subject, the method comprising: obtaininga plurality of cells harvested from the cardiac tissue of a secondsubject, wherein said second subject is an adult, wherein said cellswere have been expanded in culture to yield a population ofcardiosphere-derived cells (CDCs); wherein said CDCs are not pluripotentand are committed to differentiating into cardiac tissue; administeringbetween about 1×10⁶ and about 100×10⁶ of said CDCs said a first subject,wherein said administered CDCs generate one or more cytokines,chemokines or diffusible factors, wherein, after administration, atleast a portion of said administered CDCs engraft into the cardiactissue of said first subject; and wherein said one or more of saidgenerated cytokines, chemokines or diffusible factors or saidengraftment improves the function of said damaged cardiac tissue,thereby treating said first subject.
 20. The method of claim 19, whereinsaid method reduces the risk of producing undesired tissue growth insaid first subject as compared to treatment with embryonic cells.